Transcription factor stress-related proteins and methods of use in plants

ABSTRACT

A transgenic plant transformed by a Transcription Factor Stress-Related Protein (TFSRP) coding nucleic acid, wherein expression of the nucleic acid sequence in the plant results in increased tolerance to environmental stress as compared to a wild type variety of the plant. Also provided are agricultural products, including seeds, produced by the transgenic plants. Also provided are isolated TFSRPs, and isolated nucleic acid coding TFSRPs, and vectors and host cells containing the latter.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional application of U.S.Nonprovisional patent application Ser. No. 09/828,303 filed Apr. 6,2001, and claims the priority benefit of U.S. Provisional ApplicationSerial No. 60/196,001 filed Apr. 7, 2000, both of which are herebyincorporated in their entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates generally to nucleic acid sequencesencoding proteins that are associated with abiotic stress responses andabiotic stress tolerance in plants. In particular, this inventionrelates to nucleic acid sequences encoding proteins that confer drought,cold, and/or salt tolerance to plants.

[0004] 2. Background Art

[0005] Abiotic environmental stresses, such as drought stress, salinitystress, heat stress, and cold stress, are major limiting factors ofplant growth and productivity. Crop losses and crop yield losses ofmajor crops such as rice, maize (corn) and wheat caused by thesestresses represent a significant economic and political factor andcontribute to food shortages in many underdeveloped countries.

[0006] Plants are typically exposed during their life cycle toconditions of reduced environmental water content. Most plants haveevolved strategies to protect themselves against these conditions ofdesiccation. However, if the severity and duration of the droughtconditions are too great, the effects on plant development, growth andyield of most crop plants are profound. Furthermore, most of the cropplants are very susceptible to higher salt concentrations in the soil.Continuous exposure to drought and high salt causes major alterations inthe plant metabolism. These great changes in metabolism ultimately leadto cell death and consequently yield losses.

[0007] Developing stress-tolerant plants is a strategy that has thepotential to solve or mediate at least some of these problems. However,traditional plant breeding strategies to develop new lines of plantsthat exhibit resistance (tolerance) to these types of stresses arerelatively slow and require specific resistant lines for crossing withthe desired line. Limited germplasm resources for stress tolerance andincompatibility in crosses between distantly related plant speciesrepresent significant problems encountered in conventional breeding.Additionally, the cellular processes leading to drought, cold and salttolerance in model, drought- and/or salt-tolerant plants are complex innature and involve multiple mechanisms of cellular adaptation andnumerous metabolic pathways. This multi-component nature of stresstolerance has not only made breeding for tolerance largely unsuccessful,but has also limited the ability to genetically engineer stresstolerance plants using biotechnological methods.

[0008] Therefore, what is needed is the identification of the genes andproteins involved in these multi-component processes leading to stresstolerance. Elucidating the function of genes expressed in stresstolerant plants will not only advance our understanding of plantadaptation and tolerance to environmental stresses, but also may provideimportant information for designing new strategies for crop improvement.

[0009] One model plant used in the study of stress tolerance isArabidopsis thaliana. There are at least four differentsignal-transduction pathways leading to stress tolerance in the modelplant Arabidopsis thaliana. These pathways are under the control ofdistinct transcription factors (Shinozaki et al., 2000 Curr. Op. Pl.Biol. 3:217-23). Regulators of genes, especially transcription factors,involved in these tolerance pathways are particularly suitable forengineering tolerance into plants because a single gene can activate awhole cascade of genes leading to the tolerant phenotype. Consequently,transcription factors are important targets in the quest to identifygenes conferring stress tolerance to plants.

[0010] One transcription factor that has been identified in the priorart is the Arabidopsis thaliana transcription factor CBF (Jaglo-Ottosenet al., 1998 Science 280:104-6). Over-expression of this gene inArabidopsis conferred drought tolerance to this plant (Kasuga et al.,1999 Nature Biotech. 17:287-91). However, CBF is the only example todate of a transcription factor able to confer drought tolerance toplants upon over-expression.

[0011] Although some genes that are involved in stress responses inplants have been characterized, the characterization and cloning ofplant genes that confer stress tolerance remains largely incomplete andfragmented. For example, certain studies have indicated that drought andsalt stress in some plants may be due to additive gene effects, incontrast to other research that indicates specific genes aretranscriptionally activated in vegetative tissue of plants under osmoticstress conditions. Although it is generally assumed that stress-inducedproteins have a role in tolerance, direct evidence is still lacking, andthe functions of many stress-responsive genes are unknown.

[0012] There is a need, therefore, to identify genes expressed in stresstolerant plants that have the capacity to confer stress resistance toits host plant and to other plant species. Newly generated stresstolerant plants will have many advantages, such as increasing the rangethat crop plants can be cultivated by, for example, decreasing the waterrequirements of a plant species.

SUMMARY OF THE INVENTION

[0013] This invention fulfills in part the need to identify new, uniquetranscription factors capable of conferring stress tolerance to plantsupon over-expression. The present invention provides a transgenic plantcell transformed by a Transcription Factor Stress-Related Protein(TFSRP) coding nucleic acid, wherein expression of the nucleic acidsequence in the plant cell results in increased tolerance toenvironmental stress as compared to a wild type variety of the plantcell. Namely, described herein are the transcription factors 1) CAAT-Boxlike Binding Factor-3 (CABF-3); 2) Zinc Finger-2 (ZF-2) 3) Zinc Finger-3(ZF-3); 4) Zinc Finger-4 (ZF4); 5) Zinc Finger-5 (ZF-5); 6) AP2 SimilarFactor-2 (APS-2); 7) Sigma Factor Like Factor-1 (SFL-1); and 8) MYBFactor-1 (MYB-1), all from Physcomitrella patens.

[0014] The invention provides in some embodiments that the TFSRP andcoding nucleic acid are that found in members of the genusPhyscomitrella. In another preferred embodiment, the nucleic acid andprotein are from a Physcomitrella patens. The invention provides thatthe environmental stress can be salinity, drought, temperature, metal,chemical, pathogenic and oxidative stresses, or combinations thereof. Inpreferred embodiments, the environmental stress can be drought or coldtemperature.

[0015] The invention further provides a seed produced by a transgenicplant transformed by a TFSRP coding nucleic acid, wherein the plant istrue breeding for increased tolerance to environmental stress ascompared to a wild type variety of the plant. The invention furtherprovides a seed produced by a transgenic plant expressing a TFSRP,wherein the plant is true breeding for increased tolerance toenvironmental stress as compared to a wild type variety of the plant.

[0016] The invention further provides an agricultural product producedby any of the below-described transgenic plants, plant parts or seeds.The invention further provides an isolated TFSRP as described below. Theinvention further provides an isolated TFSRP coding nucleic acid,wherein the TFSRP coding nucleic acid codes for a TFSRP as describedbelow.

[0017] The invention further provides an isolated recombinant expressionvector comprising a TFSRP coding nucleic acid as described below,wherein expression of the vector in a host cell results in increasedtolerance to environmental stress as compared to a wild type variety ofthe host cell. The invention further provides a host cell containing thevector and a plant containing the host cell.

[0018] The invention further provides a method of producing a transgenicplant with a TFSRP coding nucleic acid, wherein expression of thenucleic acid in the plant results in increased tolerance toenvironmental stress as compared to a wild type variety of the plantcomprising: (a) transforming a plant cell with an expression vectorcomprising a TFSRP coding nucleic acid, and (b) generating from theplant cell a transgenic plant with an increased tolerance toenvironmental stress as compared to a wild type variety of the plant. Inpreferred embodiments, the TFSRP and TFSRP coding nucleic acid are asdescribed below.

[0019] The present invention further provides a method of identifying anovel TFSRP, comprising (a) raising a specific antibody response to aTFSRP, or fragment thereof, as described below; (b) screening putativeTFSRP material with the antibody, wherein specific binding of theantibody to the material indicates the presence of a potentially novelTFSRP; and (c) identifying from the bound material a novel TFSRP incomparison to known TFSRP. Alternatively, hybridization with nucleicacid probes as described below can be used to identify novel TFSRPnucleic acids.

[0020] The present invention also provides methods of modifying stresstolerance of a plant comprising, modifying the expression of a TFSRP inthe plant, wherein the TFSRP is as described below. The inventionprovides that this method can be performed such that the stresstolerance is either increased or decreased. Preferably, stress toleranceis increased in a plant via increasing expression of a TFSRP.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 shows a diagram of the plant expression vector pBPSSC022containing the super promoter driving the expression of SEQ ID NOs: 9,10, 11, 12, 13, 14, 15, and 16 (“Desired Gene”). The components are:NPTII kanamycin resistance gene (Hajdukiewicz et al. 1994 Pl. Mol. Biol.25:989-98), AtAct2-i promoter (An et al. 1996 Plant J. 10:107-21), OCS3terminator (Weigel et al. 2000 Pl. Physiol. 122: 1003-13).

[0022]FIG. 2 shows the results of a drought stress test withover-expressing PpZF-2 transgenic plants and wild-type Arabidopsislines. The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

[0023]FIG. 3 shows the results of a drought stress test withover-expressing PpZF-3 transgenic plants and wild-type Arabidopsislines. The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

[0024]FIG. 4 shows the results of a drought stress test withover-expressing PpZF-4 transgenic plants and wild-type Arabidopsislines. The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

[0025]FIG. 5 shows the results of a drought stress test withover-expressing PpZF-5 transgenic plants and wild-type Arabidopsislines. The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

[0026]FIG. 6 shows the results of a drought stress test withover-expressing PpCABF-3 transgenic plants and wild-type Arabidopsislines. The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

[0027]FIG. 7 shows the results of a drought stress test withover-expressing PpAPS-2 transgenic plants and wild-type Arabidopsislines. The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

[0028]FIG. 8 shows the results of a drought stress test withover-expressing PpSFL-1 transgenic plants and wild-type Arabidopsislines. The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

[0029]FIG. 9 shows the results of a drought stress test withover-expressing PpMYB-1 transgenic plants and wild-type Arabidopsislines. The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

[0030]FIG. 10 shows the results of a freezing stress test withover-expressing PpCABF-3 transgenic plants and wild-type Arabidopsislines. The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

[0031]FIG. 11 shows the results of a freezing stress test withover-expressing PpZF-2 transgenic plants and wild-type Arabidopsislines. The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

[0032]FIG. 12 shows the results of a freezing stress test withover-expressing PpZF-3 transgenic plants and wild-type Arabidopsislines. The transgenic lines display a tolerant phenotype. Individualtransformant lines are shown.

DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention may be understood more readily by referenceto the following detailed description of the preferred embodiments ofthe invention and the Examples included herein. However, before thepresent compounds, compositions, and methods are disclosed anddescribed, it is to be understood that this invention is not limited tospecific nucleic acids, specific polypeptides, specific cell types,specific host cells, specific conditions, or specific methods, etc., assuch may, of course, vary, and the numerous modifications and variationstherein will be apparent to those skilled in the art. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing specific embodiments only and is not intended to be limiting.In particular, the designation of the amino acid sequences as protein“Transcription Factor Stress-Related Proteins” (TFSRPs), in no waylimits the functionality of those sequences.

[0034] The present invention provides a transgenic plant celltransformed by a TFSRP coding nucleic acid, wherein expression of thenucleic acid sequence in the plant cell results in increased toleranceto environmental stress as compared to a wild type variety of the plantcell. The invention further provides transgenic plant parts andtransgenic plants containing the plant cells described herein. Alsoprovided is a plant seed produced by a transgenic plant transformed by aTFSRP coding nucleic acid, wherein the seed contains the TFSRP codingnucleic acid, and wherein the plant is true breeding for increasedtolerance to environmental stress as compared to a wild type variety ofthe plant. The invention further provides a seed produced by atransgenic plant expressing a TFSRP, wherein the seed contains theTFSRP, and wherein the plant is true breeding for increased tolerance toenvironmental stress as compared to a wild type variety of the plant.The invention also provides an agricultural product produced by any ofthe below-described transgenic plants, plant parts and plant seeds.

[0035] As used herein, the term “variety” refers to a group of plantswithin a species that share constant characters that separate them fromthe typical form and from other possible varieties within that species.While possessing at least one distinctive trait, a variety is alsocharacterized by some variation between individuals within the variety,based primarily on the Mendelian segregation of traits among the progenyof succeeding generations. A variety is considered “true breeding” for aparticular trait if it is genetically homozygous for that trait to theextent that, when the true-breeding variety is self-pollinated, asignificant amount of independent segregation of the trait among theprogeny is not observed. In the present invention, the trait arises fromthe transgenic expression of one or more DNA sequences introduced into aplant variety.

[0036] The present invention describes for the first time that thePhyscomitrella patens TFSRPs, APS-2, ZF-2, ZF-3, ZF-4, ZF-5, MYB-1,CABF-3 and SFL-1, are useful for increasing a plant's tolerance toenvironmental stress. The PpAPS-2 protein (AP2 Similar) contains aregion of similarity with the AP2 domain present in some planttranscription factors. Apetala-2 (AP2) is a homeotic gene in Arabidopsisand mutations in this gene result in the generation of flowers withoutpetals. The AP2 domain is found in not only homeotic genes in plants,but also in proteins with diverse function.

[0037] Another group of novel predicted proteins described herein arePpZF-2, PpZF-3, PpZF-4 and PpZF-5, which show sequence similarity to theZinc-Finger class of transcription factors. Zinc-finger transcriptionfactors share in common a specific secondary structure wherein a zincmolecule is sequestered by the interaction with cysteine or histidineamino acid residues. Through these “fingers,” the proteins interact withtheir specific DNA targets and regulate transcription of the targetgenes. Zinc-finger factors are associated with a multitude of biologicalphenomena. For example, in yeast zinc fingers are related with theregulation of multiple genes, e.g. genes involved in general metabolism.In plants, a zinc-finger protein, CONSTANS, is responsible fordetermining flowering time (Putterill et al. 1995 Cell 80:847-57).Sakamoto et al. (2000 Gene 248:23-32) also report the activation of thegene expression of three zinc finger proteins in Arabidopsis duringwater-stress treatments. They did not, however, present any data linkingthis increased expression with stress tolerance. Finally, Lippuner etal. (1996 JBC 271:12859-66) have reported that a particular class ofzinc-finger proteins was able to confer salt tolerance to yeast mutants,however no data showing increased salt tolerance to whole plants waspresented.

[0038] Another novel predicted protein described herein is a PpMYB-1protein that shares sequence homology with transcription factors fromthe MYB family. This group of transcription factors have the highestdegree of homology in the “MYB domain”. In addition to being involved inpigment formation in maize (Shinozaki et al. 2000. Curr. Op. Pl. Biol.3: 217-23), it has also been proposed that a MYB-containing protein isinvolved in regulating stress-related gene expression in plants. Inparticular, a MYB-containing protein, AtMYB2 has been shown to bestress-induced (PCT Application No. WO 99/16878). However, no data hasbeen presented, demonstrating that the over-expression of AtMYB2 leadsto stress tolerance in a plant.

[0039] Yet another novel predicted protein described herein is PpCABF-3,which is similar to the domain “B” of other CAAT-Box Binding Factors(Johnson and McKnight, 1989. Ann. Rev. Biochem. 58:799-840). In general,CABFs are parts of multi-component transcription activation complexesand act as general transcriptional regulators and activators. Theparticular combination of the different CABFs and other sub-units in thecomplex determines the target genes. PpCABF-3 seems to be important forthe activation of stress-related genes upon over-expression inArabidopsis thaliana. PpCABF-3 is homologous to other two CAAT-BoxBinding Factors from Physcomitrella patens, namely PpCABF-1 andPpCABF-2. Based upon a phylogenic analysis, it is believed that theseproteins belong to an exclusive class of CAAT-Box Binding proteins.

[0040] A final group of novel predicted proteins described hereinincludes the PpSFL-1 (Sigma Factor Like) protein. The SFL-1 shares ahigh degree of sequence with prokaryotic and plant chloroplast sigmafactors. Sigma factors are essential for determining promoterrecognition and consequently correct transcription initiation inprokaryotes as well as in chloroplasts. Chloroplasts are a major targetfor engineering stress tolerance, since these organelles are heavilyimpaired during stress conditions. Attenuation of chloroplast damage canlead to increased stress tolerance in plants.

[0041] Accordingly, the present invention provides isolated TFSRPsselected from the group consisting of APS-2, ZF-2, ZF-3, ZF-4, ZF-5,MYB-1, CABF-3, SFL-1 and homologs thereof. In preferred embodiments, theTFSRP is selected from 1) a AP2 Similar-2 (APS-2) protein as defined inSEQ ID NO:17; 2) a Zinc-Finger Factor-2 (ZF-2) protein as defined in SEQID NO:18; 3) a Zinc-Finger Factor-3 (ZF-3) protein as defined in SEQ IDNO:19; 4) a Zinc-Finger Factor-4 (ZF-4) protein as defined in SEQ IDNO:20; 5) a Zinc-Finger Factor-5 (ZF-5) protein as defined in SEQ IDNO:21; 6) a MYB-1 (MYB-1) protein as defined in SEQ ID NO:22; 7) aCAAT-Box Binding Factor-3 (CABF-3) protein as defined in SEQ ID NO:23;8) a Sigma Factor Like (SFL-1) protein as defined in SEQ ID NO:24, andhomologs and orthologs thereof. Homologs and orthologs of the amino acidsequences are defined below.

[0042] The TFSRPs of the present invention are preferably produced byrecombinant DNA techniques. For example, a nucleic acid moleculeencoding the protein is cloned into an expression vector (as describedbelow), the expression vector is introduced into a host cell (asdescribed below) and the TFSRP is expressed in the host cell. The TFSRPcan then be isolated from the cells by an appropriate purificationscheme using standard protein purification techniques. Alternative torecombinant expression, a TFSRP polypeptide, or peptide can besynthesized chemically using standard peptide synthesis techniques.Moreover, native TFSRP can be isolated from cells (e.g., Physcomitrellapatens), for example using an anti-TFSRP antibody, which can be producedby standard techniques utilizing a TFSRP or fragment thereof.

[0043] The invention further provides an isolated TFSRP coding nucleicacid. The present invention includes TFSRP coding nucleic acids thatencode TFSRPs as described herein. In preferred embodiments, the TFSRPcoding nucleic acid is selected from 1) a AP2 Similar-2 (APS-2) nucleicacid as defined in SEQ ID NO:9; 2) a Zinc-Finger Factor-2 (ZF-2) nucleicacid as defined in SEQ ID NO:10; 3) a Zinc-Finger Factor-3 (ZF-3)nucleic acid as defined in SEQ ID NO:11; 4) a Zinc-Finger Factor-4 (ZFA)nucleic acid as defined in SEQ ID NO:12; 5) a Zinc-Finger Factor-5(ZF-5) nucleic acid as defined in SEQ ID NO:13; 6) a MYB-1 nucleic acidas defined in SEQ ID NO:14; 7) a CAAT-Box Binding Factor-3 (CABF-3)nucleic acid as defined in SEQ ID NO:15; 8) a Sigma Factor Like (SFL-1)nucleic acid as defined in SEQ ID NO:16 and homologs and orthologsthereof. Homologs and orthologs of the nucleotide sequences are definedbelow. In one preferred embodiment, the nucleic acid and protein areisolated from the plant genus Physcomitrella. In another preferredembodiment, the nucleic acid and protein are from a Physcomitrellapatens (P. patens) plant.

[0044] As used herein, the term “environmental stress” refers to anysub-optimal growing condition and includes, but is not limited to,sub-optimal conditions associated with salinity, drought, temperature,metal, chemical, pathogenic and oxidative stresses, or combinationsthereof. In preferred embodiments, the environmental stress can besalinity, drought, or temperature, or combinations thereof, and inparticular, can be high salinity, low water content or low temperature.It is also to be understood that as used in the specification and in theclaims, “a” or “an” can mean one or more, depending upon the context inwhich it is used. Thus, for example, reference to “a cell” can mean thatat least one cell can be utilized.

[0045] As also used herein, the terms “nucleic acid” and “nucleic acidmolecule” are intended to include DNA molecules (e.g., cDNA or genomicDNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNAgenerated using nucleotide analogs. This term also encompassesuntranslated sequence located at both the 3′ and 5′ ends of the codingregion of the gene: at least about 1000 nucleotides of sequence upstreamfrom the 5′ end of the coding region and at least about 200 nucleotidesof sequence downstream from the 3′ end of the coding region of the gene.The nucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

[0046] An “isolated” nucleic acid molecule is one that is substantiallyseparated from other nucleic acid molecules which are present in thenatural source of the nucleic acid. Preferably, an “isolated” nucleicacid is free of some of the sequences which naturally flank the nucleicacid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid)in the genomic DNA of the organism from which the nucleic acid isderived. For example, in various embodiments, the isolated TFSRP nucleicacid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb,0.5 kb or 0.1 kb of nucleotide sequences which naturally flank thenucleic acid molecule in genomic DNA of the cell from which the nucleicacid is derived (e.g., a Physcomitrella patens cell). Moreover, an“isolated” nucleic acid molecule, such as a cDNA molecule, can be freefrom some of the other cellular material with which it is naturallyassociated, or culture medium when produced by recombinant techniques,or chemical precursors or other chemicals when chemically synthesized.

[0047] A nucleic acid molecule of the present invention, e.g., a nucleicacid molecule having a nucleotide sequence of SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, or a portion thereof, can be isolated using standardmolecular biology techniques and the sequence information providedherein. For example, a P. patens TFSRP cDNA can be isolated from a P.patens library using all or portion of one of the sequences of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7 or SEQ ID NO:8. Moreover, a nucleic acid moleculeencompassing all or a portion of one of the sequences of SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7 and SEQ ID NO:8 can be isolated by the polymerase chain reactionusing oligonucleotide primers designed based upon this sequence. Forexample, mRNA can be isolated from plant cells (e.g., by theguanidinium-thiocyanate extraction procedure of Chirgwin et al., 1979Biochemistry 18:5294-5299) and cDNA can be prepared using reversetranscriptase (e.g., Moloney MLV reverse transcriptase, available fromGibco/BRL, Bethesda, Md.; or AMV reverse transcriptase, available fromSeikagaku America, Inc., St. Petersburg, Fla.). Syntheticoligonucleotide primers for polymerase chain reaction amplification canbe designed based upon one of the nucleotide sequences shown in SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7 and SEQ ID NO:8. A nucleic acid molecule of the inventioncan be amplified using cDNA or, alternatively, genomic DNA, as atemplate and appropriate oligonucleotide primers according to standardPCR amplification techniques. The nucleic acid molecule so amplified canbe cloned into an appropriate vector and characterized by DNA sequenceanalysis. Furthermore, oligonucleotides corresponding to a TFSRPnucleotide sequence can be prepared by standard synthetic techniques,e.g., using an automated DNA synthesizer.

[0048] In a preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises one of the nucleotide sequences shown in SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15 and SEQ ID NO:16. These cDNAs comprise sequencesencoding the TFSRPs (i.e., the “coding region”, indicated in Table 1),as well as 5′ untranslated sequences and 3′ untranslated sequences. Itis to be understood that SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16comprise both coding regions and 5′ and 3′ untranslated regions.Alternatively, the nucleic acid molecules of the present invention cancomprise only the coding region of any of the sequences in SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15 or SEQ ID NO:16 or can contain whole genomic fragmentsisolated from genomic DNA. A coding region of these sequences isindicated as “ORF position”. The present invention also includes TFSRPcoding nucleic acids that encode TFSRPs as described herein. Preferredis a TFSRP coding nucleic acid that encodes a TFSRP selected from thegroup consisting of, APS-2 (SEQ ID NO:17), ZF-2 (SEQ ID NO:18), ZF-3(SEQ ID NO:19), ZF-4 (SEQ ID NO:20), ZF-5 (SEQ ID NO:21), MYB-1 (SEQ IDNO:22), CABF-3 (SEQ ID NO:23) and SFL-1 (SEQ ID NO:24).

[0049] Moreover, the nucleic acid molecule of the invention can compriseonly a portion of the coding region of one of the sequences in SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15 and SEQ ID NO:16, for example, a fragment which canbe used as a probe or primer or a fragment encoding a biologicallyactive portion of a TFSRP. The nucleotide sequences determined from thecloning of the TFSRP genes from P. patens allow for the generation ofprobes and primers designed for use in identifying and/or cloning TFSRPhomologs in other cell types and organisms, as well as TFSRP homologsfrom other mosses and related species.

[0050] Portions of proteins encoded by the TFSRP nucleic acid moleculesof the invention are preferably biologically active portions of one ofthe TFSRPs described herein. As used herein, the term “biologicallyactive portion of a TFSRP is intended to include a portion, e.g., adomain/motif, of a TFSRP that participates in a stress toleranceresponse in a plant, has an activity as set forth in Table 1, orparticipates in the transcription of a protein involved in a stresstolerance response in a plant. To determine whether a TFSRP, or abiologically active portion thereof, can participate in transcription ofa protein involved in a stress tolerance response in a plant, or whetherrepression of a TFSRP results in increased stress tolerance in a plant,a stress analysis of a plant comprising the TFSRP may be performed. Suchanalysis methods are well known to those skilled in the art, as detailedin Example 7. More specifically, nucleic acid fragments encodingbiologically active portions of a TFSRP can be prepared by isolating aportion of one of the sequences in SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ IDNO:24, expressing the encoded portion of the TFSRP or peptide (e.g., byrecombinant expression in vitro) and assessing the activity of theencoded portion of the TFSRP or peptide.

[0051] Biologically active portions of a TFSRP are encompassed by thepresent invention and include peptides comprising amino acid sequencesderived from the amino acid sequence of a TFSRP, e.g., an amino acidsequence of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24, or the amino acidsequence of a protein homologous to a TFSRP, which include fewer aminoacids than a full length TFSRP or the full length protein which ishomologous to a TFSRP, and exhibit at least one activity of a TFSRP.Typically, biologically active portions (e.g., peptides which are, forexample, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or moreamino acids in length) comprise a domain or motif with at least oneactivity of a TFSRP. Moreover, other biologically active portions inwhich other regions of the protein are deleted, can be prepared byrecombinant techniques and evaluated for one or more of the activitiesdescribed herein. Preferably, the biologically active portions of aTFSRP include one or more selected domains/motifs or portions thereofhaving biological activity.

[0052] The invention also provides TFSRP chimeric or fusion proteins. Asused herein, a TFSRP “chimeric protein” or “fusion protein” comprises aTFSRP polypeptide operatively linked to a non-TFSRP polypeptide. A TFSRPpolypeptide refers to a polypeptide having an amino acid sequencecorresponding to a TFSRP, whereas a non-TFSRP polypeptide refers to apolypeptide having an amino acid sequence corresponding to a proteinwhich is not substantially homologous to the TFSRP, e.g., a protein thatis different from the TFSRP and is derived from the same or a differentorganism. Within the fusion protein, the term “operatively linked” isintended to indicate that the TFSRP polypeptide and the non-TFSRPpolypeptide are fused to each other so that both sequences fulfill theproposed function attributed to the sequence used. The non-TFSRPpolypeptide can be fused to the N-terminus or C-terminus of the TFSRPpolypeptide. For example, in one embodiment, the fusion protein is aGST-TFSRP fusion protein in which the TFSRP sequences are fused to theC-terminus of the GST sequences. Such fusion proteins can facilitate thepurification of recombinant TFSRPs. In another embodiment, the fusionprotein is a TFSRP containing a heterologous signal sequence at itsN-terminus. In certain host cells (e.g., mammalian host cells),expression and/or secretion of a TFSRP can be increased through use of aheterologous signal sequence.

[0053] Preferably, a TFSRP chimeric or fusion protein of the inventionis produced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and re-amplified togenerate a chimeric gene sequence (see, for example, Current Protocolsin Molecular Biology, Eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). A TFSRPencoding nucleic acid can be cloned into such an expression vector suchthat the fusion moiety is linked in-frame to the TFSRP.

[0054] In addition to fragments and fusion proteins of the TFSRPsdescribed herein, the present invention includes homologs and analogs ofnaturally occurring TFSRPs and TFSRP encoding nucleic acids in a plant.“Homologs” are defined herein as two nucleic acids or proteins that havesimilar, or “homologous”, nucleotide or amino acid sequences,respectively. Homologs include allelic variants, orthologs, paralogs,agonists and antagonists of TFSRPs as defined hereafter. The term“homolog” further encompasses nucleic acid molecules that differ fromone of the nucleotide sequences shown in SEQ ID NO:9, SEQ ID NO:10, SEQID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQID NO:16 (and portions thereof) due to degeneracy of the genetic codeand thus encode the same TFSRP as that encoded by the nucleotidesequences shown in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16. As usedherein a “naturally occurring” TFSRP refers to a TFSRP amino acidsequence that occurs in nature. Preferably, a naturally occurring TFSRPcomprises an amino acid sequence selected from the group consisting ofSEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24.

[0055] An agonist of the TFSRP can retain substantially the same, or asubset, of the biological activities of the TFSRP. An antagonist of theTFSRP can inhibit one or more of the activities of the naturallyoccurring form of the TFSRP. For example, the TFSRP antagonist cancompetitively bind to a downstream or upstream member of the cellmembrane component metabolic cascade that includes the TFSRP, or bind toa TFSRP that mediates transport of compounds across such membranes,thereby preventing translocation from taking place.

[0056] Nucleic acid molecules corresponding to natural allelic variantsand analogs, orthologs and paralogs of a TFSRP cDNA can be isolatedbased on their identity to the Physcomitrella patens TFSRP nucleic acidsdescribed herein using TFSRP cDNAs, or a portion thereof, as ahybridization probe according to standard hybridization techniques understringent hybridization conditions. In an alternative embodiment,homologs of the TFSRP can be identified by screening combinatoriallibraries of mutants, e.g., truncation mutants, of the TFSRP for TFSRPagonist or antagonist activity. In one embodiment, a variegated libraryof TFSRP variants is generated by combinatorial mutagenesis at thenucleic acid level and is encoded by a variegated gene library. Avariegated library of TFSRP variants can be produced by, for example,enzymatically ligating a mixture of synthetic oligonucleotides into genesequences such that a degenerate set of potential TFSRP sequences isexpressible as individual polypeptides, or alternatively, as a set oflarger fusion proteins (e.g., for phage display) containing the set ofTFSRP sequences therein. There are a variety of methods that can be usedto produce libraries of potential TFSRP homologs from a degenerateoligonucleotide sequence. Chemical synthesis of a degenerate genesequence can be performed in an automatic DNA synthesizer, and thesynthetic gene is then ligated into an appropriate expression vector.Use of a degenerate set of genes allows for the provision, in onemixture, of all of the sequences encoding the desired set of potentialTFSRP sequences. Methods for synthesizing degenerate oligonucleotidesare known in the art (see, e.g., Narang, S. A., 1983 Tetrahedron 39:3;Itakura et al., 1984 Annu. Rev. Biochem. 53:323; Itakura et al., 1984Science 198:1056; Ike et al., 1983 Nucleic Acid Res. 11:477).

[0057] In addition, libraries of fragments of the TFSRP coding regionscan be used to generate a variegated population of TFSRP fragments forscreening and subsequent selection of homologs of a TFSRP. In oneembodiment, a library of coding sequence fragments can be generated bytreating a double stranded PCR fragment of a TFSRP coding sequence witha nuclease under conditions wherein nicking occurs only about once permolecule, denaturing the double stranded DNA, renaturing the DNA to formdouble stranded DNA, which can include sense/antisense pairs fromdifferent nicked products, removing single stranded portions fromreformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the TFSRP.

[0058] Several techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations ortruncation, and for screening cDNA libraries for gene products having aselected property. Such techniques are adaptable for rapid screening ofthe gene libraries generated by the combinatorial mutagenesis of TFSRPhomologs. The most widely used techniques, which are amenable to highthrough-put analysis, for screening large gene libraries typicallyinclude cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates isolation of the vectorencoding the gene whose product was detected. Recursive ensemblemutagenesis (REM), a new technique that enhances the frequency offunctional mutants in the libraries, can be used in combination with thescreening assays to identify TFSRP homologs (Arkin and Yourvan, 1992PNAS 89:7811-7815; Delgrave et al., 1993 Protein Engineering6(3):327-331). In another embodiment, cell based assays can be exploitedto analyze a variegated TFSRP library, using methods well known in theart. The present invention further provides a method of identifying anovel TFSRP, comprising (a) raising a specific antibody response to aTFSRP, or a fragment thereof, as described above; (b) screening putativeTFSRP material with the antibody, wherein specific binding of theantibody to the material indicates the presence of a potentially novelTFSRP; and (c) analyzing the bound material in comparison to knownTFSRP, to determine its novelty.

[0059] To determine the percent homology of two amino acid sequences(e.g., one of the sequences of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24and a mutant form thereof), the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in the sequence of oneprotein or nucleic acid for optimal alignment with the other protein ornucleic acid). The amino acid residues at corresponding amino acidpositions are then compared. When a position in one sequence (e.g., oneof the sequences of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ BDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24) isoccupied by the same amino acid residue as the corresponding position inthe other sequence (e.g., a mutant form of the sequence selected fromthe polypeptide of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ BD NO:24), thenthe molecules are homologous at that position (i.e., as used hereinamino acid or nucleic acid “homology” is equivalent to amino acid ornucleic acid “identity”). The same type of comparison can be madebetween two nucleic acid sequences.

[0060] The percent homology between the two sequences is a function ofthe number of identical positions shared by the sequences (i.e., %homology=numbers of identical positions/total numbers of positions×100).Preferably, the amino acid sequences included in the present inventionare at least about 50-60%, preferably at least about 60-70%, and morepreferably at least about 70-80%, 80-90%, 90-95%, and most preferably atleast about 96%, 97%, 98%, 99% or more homologous to an entire aminoacid sequence shown in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24. In yetanother embodiment, at least about 50-60%, preferably at least about60-70%, and more preferably at least about 70-80%, 80-90%, 90-95%, andmost preferably at least about 96%, 97%, 98%, 99% or more homologous toan entire amino acid sequence encoded by a nucleic acid sequence shownin SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13,SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16. In other embodiments, thepreferable length of sequence comparison for proteins is at least 15amino acid residues, more preferably at least 25 amino acid residues,and most preferably at least 35 amino acid residues.

[0061] In another preferred embodiment, an isolated nucleic acidmolecule of the invention comprises a nucleotide sequence which is atleast about 50-60%, preferably at least about 60-70%, more preferably atleast about 70-80%, 80-90%, or 90-95%, and even more preferably at leastabout 95%, 96%, 97%, 98%, 99% or more homologous to a nucleotidesequence shown in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12,SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16, or a portionthereof. The preferable length of sequence comparison for nucleic acidsis at least 75 nucleotides, more preferably at least 100 nucleotides andmost preferably the entire coding region.

[0062] It is also preferable that the homologous nucleic acid moleculeof the invention encodes a protein or portion thereof which includes anamino acid sequence which is sufficiently homologous to an amino acidsequence of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24 such that theprotein or portion thereof maintains the same or a similar function asthe amino acid sequence to which it is compared. Functions of the TFSRPamino acid sequences of the present invention include the ability toparticipate in a stress tolerance response in a plant, or moreparticularly, to participate in the transcription of a protein involvedin a stress tolerance response in a Physcomitrella patens plant.Examples of such activities are described in Table 1.

[0063] In addition to the above described methods, a determination ofthe percent homology between two sequences can be accomplished using amathematical algorithm. A preferred, non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul (1990 Proc. Natl. Acad. Sci. USA90:5873-5877). Such an algorithm is incorporated into the NBLAST andXBLAST programs of Altschul, et al. (1990 J. Mol. Biol. 215:403-410).

[0064] BLAST nucleic acid searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleic acid sequenceshomologous to the TFSRP nucleic acid molecules of the invention.Additionally, BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to TFSRPs of the present invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al. (1997 Nucleic Acids Res. 25:3389-3402).When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) can be used.Another preferred non-limiting example of a mathematical algorithmutilized for the comparison of sequences is the algorithm of Myers andMiller (CABIOS 1989). Such an algorithm is incorporated into the ALIGNprogram (version 2.0) that is part of the GCG sequence alignmentsoftware package. When utilizing the ALIGN program for comparing aminoacid sequences, a PAM120 weight residue table, a gap length penalty of12 and a gap penalty of 4 can be used to obtain amino acid sequenceshomologous to the TFSRPs of the present invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al. (1997 Nucleic Acids Res. 25:3389-3402).When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) can be used.Another preferred non-limiting example of a mathematical algorithmutilized for the comparison of sequences is the algorithm of Myers andMiller (CABIOS 1989). Such an algorithm is incorporated into the ALIGNprogram (version 2.0) that is part of the GCG sequence alignmentsoftware package. When utilizing the ALIGN program for comparing aminoacid sequences, a PAM120 weight residue table, a gap length penalty of12 and a gap penalty of 4 can be used.

[0065] Finally, homology between nucleic acid sequences can also bedetermined using hybridization techniques known to those of skill in theart. Accordingly, an isolated nucleic acid molecule of the inventioncomprises a nucleotide sequence which hybridizes, e.g., hybridizes understringent conditions, to one of the nucleotide sequences shown in SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15 and SEQ ID NO:16, or a portion thereof. Moreparticularly, an isolated nucleic acid molecule of the invention is atleast 15 nucleotides in length and hybridizes under stringent conditionsto the nucleic acid molecule comprising a nucleotide sequence of SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15 or SEQ ID NO:16. In other embodiments, the nucleicacid is at least 30, 50, 100, 250 or more nucleotides in length.

[0066] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% homologous to each othertypically remain hybridized to each other. Preferably, the conditionsare such that sequences at least about 65%, more preferably at leastabout 70%, and even more preferably at least about 75% or morehomologous to each other typically remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, 6.3.1-6.3.6, John Wiley& Sons, N.Y. (1989). A preferred, non-limiting example of stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50-65° C. Preferably, an isolated nucleic acidmolecule of the invention that hybridizes under stringent conditions toa sequence of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16 corresponds to anaturally occurring nucleic acid molecule. As used herein, a “naturallyoccurring” nucleic acid molecule refers to an RNA or DNA molecule havinga nucleotide sequence that occurs in nature (e.g., encodes a naturalprotein). In one embodiment, the nucleic acid encodes a naturallyoccurring Physcomitrella patens TFSRP.

[0067] Using the above-described methods, and others known to those ofskill in the art, one of ordinary skill in the art can isolate homologsof the TFSRPs comprising amino acid sequences shown in SEQ ID NO:17, SEQID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23 or SEQ ID NO:24 and the TFSRP nucleic acids comprising thenucleotide sequences shown in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16.One subset of these homologs are allelic variants. As used herein, theterm “allelic variant” refers to a nucleotide sequence containingpolymorphisms that lead to changes in the amino acid sequences of aTFSRP and that exist within a natural population (e.g., a plant speciesor variety). Such natural allelic variations can typically result in1-5% variance in a TFSRP nucleic acid. Allelic variants can beidentified by sequencing the nucleic acid sequence of interest in anumber of different plants, which can be readily carried out by usinghybridization probes to identify the same TFSRP genetic locus in thoseplants. Any and all such nucleic acid variations and resulting aminoacid polymorphisms or variations in a TFSRP that are the result ofnatural allelic variation and that do not alter the functional activityof a TFSRP, are intended to be within the scope of the invention.

[0068] Moreover, nucleic acid molecules encoding TFSRPs from the same orother species such as TFSRP analogs, orthologs and paralogs, areintended to be within the scope of the present invention. As usedherein, the term “analogs” refers to two nucleic acids that have thesame or similar function, but that have evolved separately in unrelatedorganisms. As used herein, the term “orthologs” refers to two nucleicacids from different species, but that have evolved from a commonancestral gene by speciation. Normally, orthologs encode proteins havingthe same or similar functions. As also used herein, the term “paralogs”refers to two nucleic acids that are related by duplication within agenome. Paralogs usually have different functions, but these functionsmay be related (Tatusov, R. L. et al. 1997 Science 278(5338):631-637).Analogs, orthologs and paralogs of a naturally occurring TFSRP candiffer from the naturally occurring TFSRP by post-translationalmodifications, by amino acid sequence differences, or by both.Post-translational modifications include in vivo and in vitro chemicalderivatization of polypeptides, e.g., acetylation, carboxylation,phosphorylation, or glycosylation, and such modifications may occurduring polypeptide synthesis or processing or following treatment withisolated modifying enzymes. In particular, orthologs of the inventionwill generally exhibit at least 80-85%, more preferably 90%, and mostpreferably 95%, 96%, 97%, 98% or even 99% identity or homology with allor part of a naturally occurring TFSRP amino acid sequence and willexhibit a function similar to a TFSRP. Orthologs of the presentinvention are also preferably capable of participating in the stressresponse in plants. In one embodiment, the TFSRP orthologs maintain theability to participate in the metabolism of compounds necessary for theconstruction of cellular membranes in Physcomitrella patens, or in thetransport of molecules across these membranes.

[0069] In addition to naturally-occurring variants of a TFSRP sequencethat may exist in the population, the skilled artisan will furtherappreciate that changes can be introduced by mutation into a nucleotidesequence, such as the sequences of SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:1, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ IDNO:16, thereby leading to changes in the amino acid sequence of theencoded TFSRP, without altering the functional ability of the TFSRP. Forexample, nucleotide substitutions leading to amino acid substitutions at“non-essential” amino acid residues can be made in the proteinsincluding a sequence of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ BD NO:16. A“non-essential” amino acid residue is a residue that can be altered fromthe wild-type sequence of one of the TFSRPs without altering theactivity of said TFSRP, whereas an “essential” amino acid residue isrequired for TFSRP activity. Other amino acid residues, however, (e.g.,those that are not conserved or only semi-conserved in the domain havingTFSRP activity) may not be essential for activity and thus are likely tobe amenable to alteration without altering TFSRP activity.

[0070] Accordingly, another aspect of the invention pertains to nucleicacid molecules encoding TFSRPs that contain changes in amino acidresidues that are not essential for TFSRP activity. Such TFSRPs differin amino acid sequence from a sequence contained in SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23 or SEQ ID NO:24, yet retain at least one of the TFSRP activitiesdescribed herein. In one embodiment, the isolated nucleic acid moleculecomprises a nucleotide sequence encoding a protein, wherein the proteincomprises an amino acid sequence at least about 50% homologous to anamino acid sequence of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24.Preferably, the protein encoded by the nucleic acid molecule is at leastabout 50-60% homologous to one of the sequences of SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23 or SEQ ID NO:24, more preferably at least about 60-70% homologousto one of the sequences of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24, evenmore preferably at least about 70-80%, 80-90%, 90-95% homologous to oneof the sequences of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24, andmost preferably at least about 96%, 97%, 98%, or 99% homologous to oneof the sequences of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24. Thepreferred TFSRP homologs of the present invention are preferably capableof participating in the stress tolerance response in a plant, or moreparticularly, participating in the transcription of a protein involvedin a stress tolerance response in a Physcomitrella patens plant, or haveone or more activities set forth in Table 1.

[0071] An isolated nucleic acid molecule encoding a TFSRP homologous toa protein sequence of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24 can becreated by introducing one or more nucleotide substitutions, additionsor deletions into a nucleotide sequence of SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 orSEQ ID NO:16 such that one or more amino acid substitutions, additionsor deletions are introduced into the encoded protein. Mutations can beintroduced into one of the sequences of SEQ ID NO:9, SEQ ID NO:10, SEQID NO:1, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQID NO:16 by standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis. Preferably, conservative amino acidsubstitutions are made at one or more predicted non-essential amino acidresidues. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain.

[0072] Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a TFSRP is preferablyreplaced with another amino acid residue from the same side chainfamily. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of a TFSRP coding sequence, suchas by saturation mutagenesis, and the resultant mutants can be screenedfor a TFSRP activity described herein to identify mutants that retainTFSRP activity. Following mutagenesis of one of the sequences of SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15 and SEQ ID NO:16, the encoded protein can beexpressed recombinantly and the activity of the protein can bedetermined by analyzing the stress tolerance of a plant expressing theprotein as described in Example 7.

[0073] In addition to the nucleic acid molecules encoding the TFSRPsdescribed above, another aspect of the invention pertains to isolatednucleic acid molecules that are antisense thereto. An “antisense”nucleic acid comprises a nucleotide sequence that is complementary to a“sense” nucleic acid encoding a protein, e.g., complementary to thecoding strand of a double-stranded cDNA molecule or complementary to anmRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bondto a sense nucleic acid. The antisense nucleic acid can be complementaryto an entire TFSRP coding strand, or to only a portion thereof. In oneembodiment, an antisense nucleic acid molecule is antisense to a “codingregion” of the coding strand of a nucleotide sequence encoding a TFSRP.The term “coding region” refers to the region of the nucleotide sequencecomprising codons that are translated into amino acid residues (e.g.,the entire coding region of , , , comprises nucleotides 1 to . . . ). Inanother embodiment, the antisense nucleic acid molecule is antisense toa “noncoding region” of the coding strand of a nucleotide sequenceencoding a TFSRP. The term “noncoding region” refers to 5′ and 3′sequences that flank the coding region that are not translated intoamino acids (i.e., also referred to as 5′ and 3′ untranslated regions).

[0074] In a preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofone of the nucleotide sequences shown in SEQ ID NO:9, SEQ ID NO:10, SEQID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQID NO:16, or a portion thereof. A nucleic acid molecule that iscomplementary to one of the nucleotide sequences shown in SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15 and SEQ ID NO:16 is one which is sufficiently complementaryto one of the nucleotide sequences shown in SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 andSEQ ID NO:16 such that it can hybridize to one of the nucleotidesequences shown in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:1, SEQ ID NO:12,SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, therebyforming a stable duplex.

[0075] Given the coding strand sequences encoding the TFSRPs disclosedherein (e.g., the sequences set forth in SEQ ID NO:9, SEQ ID NO:10, SEQID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQID NO:16), antisense nucleic acids of the invention can be designedaccording to the rules of Watson and Crick base pairing. The antisensenucleic acid molecule can be complementary to the entire coding regionof TFSRP mRNA, but more preferably is an oligonucleotide which isantisense to only a portion of the coding or noncoding region of TFSRPmRNA. For example, the antisense oligonucleotide can be complementary tothe region surrounding the translation start site of TFSRP mRNA. Anantisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25,30, 35, 40, 45 or 50 nucleotides in length.

[0076] An antisense nucleic acid of the invention can be constructedusing chemical synthesis and enzymatic ligation reactions usingprocedures known in the art. For example, an antisense nucleic acid(e.g., an antisense oligonucleotide) can be chemically synthesized usingnaturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used. Examples of modifiednucleotides which can be used to generate the antisense nucleic acidinclude 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

[0077] The antisense nucleic acid molecules of the invention aretypically administered to a cell or generated in situ such that theyhybridize with or bind to cellular mRNA and/or genomic DNA encoding aTFSRP to thereby inhibit expression of the protein, e.g., by inhibitingtranscription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. The antisense molecule can be modified such that itspecifically binds to a receptor or an antigen expressed on a selectedcell surface, e.g., by linking the antisense nucleic acid molecule to apeptide or an antibody which binds to a cell surface receptor orantigen. The antisense nucleic acid molecule can also be delivered tocells using the vectors described herein. To achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong prokaryotic, viral, or eukaryotic (includingplant) promoter are preferred.

[0078] In yet another embodiment, the antisense nucleic acid molecule ofthe invention is an α-anomeric nucleic acid molecule. An α-anomericnucleic acid molecule forms specific double-stranded hybrids withcomplementary RNA in which, contrary to the usual β-units, the strandsrun parallel to each other (Gaultier et al., 1987 Nucleic Acids. Res.15:6625-6641). The antisense nucleic acid molecule can also comprise a2′—O— methylribonucleotide (Inoue et al., 1987 Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., 1987 FEBSLett. 215:327-330).

[0079] In still another embodiment, an antisense nucleic acid of theinvention is a ribozyme. Ribozymes are catalytic RNA molecules withribonuclease activity which are capable of cleaving a single-strandednucleic acid, such as an mRNA, to which they have a complementaryregion. Thus, ribozymes (e.g., hammerhead ribozymes described inHaselhoff and Gerlach, 1988 Nature 334:585-591) can be used tocatalytically cleave TFSRP mRNA transcripts to thereby inhibittranslation of TFSRP mRNA. A ribozyme having specificity for aTFSRP-encoding nucleic acid can be designed based upon the nucleotidesequence of a TFSRP cDNA, as disclosed herein (i.e., SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15 or SEQ ID NO:16) or on the basis of a heterologous sequence to beisolated according to methods taught in this invention. For example, aderivative of a Tetrahymena L-19 IVS RNA can be constructed in which thenucleotide sequence of the active site is complementary to thenucleotide sequence to be cleaved in a TFSRP-encoding mRNA. See, e.g.,Cech et al. U.S. Pat. No. 4,987,071 and Cech et al. U.S. Pat. No.5,116,742. Alternatively, TFSRP mRNA can be used to select a catalyticRNA having a specific ribonuclease activity from a pool of RNAmolecules. See, e.g., Bartel, D. and Szostak, J. W., 1993 Science261:1411-1418.

[0080] Alternatively, TFSRP gene expression can be inhibited bytargeting nucleotide sequences complementary to the regulatory region ofa TFSRP nucleotide sequence (e.g., a TFSRP promoter and/or enhancer) toform triple helical structures that prevent transcription of a TFSRPgene in target cells. See generally, Helene, C., 1991 Anticancer DrugDes. 6(6):569-84; Helene, C. et al., 1992 Ann. N.Y. Acad. Sci.660:27-36; and Maher, L. J., 1992 Bioassays 14(12):807-15.

[0081] In addition to the TFSRP nucleic acids and proteins describedabove, the present invention encompasses these nucleic acids andproteins attached to a moiety. These moieties include, but are notlimited to, detection moieties, hybridization moieties, purificationmoieties, delivery moieties, reaction moieties, binding moieties, andthe like. One typical group of nucleic acids attached to a moiety areprobes and primers. The probe/primer typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, preferably about 25, more preferably about 40, 50 or 75consecutive nucleotides of a sense strand of one of the sequences setforth in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, an anti-sensesequence of one of the sequences set forth in SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 andSEQ ID NO:16, or naturally occurring mutants thereof. Primers based on anucleotide sequence of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16 can beused in PCR reactions to clone TFSRP homologs. Probes based on the TFSRPnucleotide sequences can be used to detect transcripts or genomicsequences encoding the same or homologous proteins. In preferredembodiments, the probe further comprises a label group attached thereto,e.g. the label group can be a radioisotope, a fluorescent compound, anenzyme, or an enzyme co-factor. Such probes can be used as a part of agenomic marker test kit for identifying cells which express a TFSRP,such as by measuring a level of a TFSRP-encoding nucleic acid, in asample of cells, e.g., detecting TFSRP mRNA levels or determiningwhether a genomic TFSRP gene has been mutated or deleted.

[0082] In particular, a useful method to ascertain the level oftranscription of the gene (an indicator of the amount of mRNA availablefor translation to the gene product) is to perform a Northern blot (forreference see, for example, Ausubel et al., 1988 Current Protocols inMolecular Biology, Wiley: New York). This information at least partiallydemonstrates the degree of transcription of the transformed gene. Totalcellular RNA can be prepared from cells, tissues or organs by severalmethods, all well-known in the art, such as that described in Bormann,E. R. et al., 1992 Mol. Microbiol. 6:317-326. To assess the presence orrelative quantity of protein translated from this mRNA, standardtechniques, such as a Western blot, may be employed. These techniquesare well known to one of ordinary skill in the art. (See, for example,Ausubel et al., 1988 Current Protocols in Molecular Biology, Wiley: NewYork).

[0083] The invention further provides an isolated recombinant expressionvector comprising a TFSRP nucleic acid as described above, whereinexpression of the vector in a host cell results in increased toleranceto environmental stress as compared to a wild type variety of the hostcell. As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

[0084] The recombinant expression vectors of the invention comprise anucleic acid of the invention in a form suitable for expression of thenucleic acid in a host cell, which means that the recombinant expressionvectors include one or more regulatory sequences, selected on the basisof the host cells to be used for expression, which is operatively linkedto the nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory sequence(s)in a manner which allows for expression of the nucleotide sequence(e.g., in an in vitro transcription/translation system or in a host cellwhen the vector is introduced into the host cell). The term “regulatorysequence” is intended to include promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Suchregulatory sequences are described, for example, in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) or see: Gruber and Crosby, in: Methods in PlantMolecular Biology and Biotechnology, eds. Glick and Thompson, Chapter 7,89-108, CRC Press: Boca Raton, Fla., including the references therein.Regulatory sequences include those that direct constitutive expressionof a nucleotide sequence in many types of host cells and those thatdirect expression of the nucleotide sequence only in certain host cellsor under certain conditions. It will be appreciated by those skilled inthe art that the design of the expression vector can depend on suchfactors as the choice of the host cell to be transformed, the level ofexpression of protein desired, etc. The expression vectors of theinvention can be introduced into host cells to thereby produce proteinsor peptides, including fusion proteins or peptides, encoded by nucleicacids as described herein (e.g., TFSRPs, mutant forms of TFSRPs, fusionproteins, etc.).

[0085] The recombinant expression vectors of the invention can bedesigned for expression of TFSRPs in prokaryotic or eukaryotic cells.For example, TFSRP genes can be expressed in bacterial cells such as C.glutamicum, insect cells (using baculovirus expression vectors), yeastand other fungal cells (see Romanos, M. A. et al., 1992 Foreign geneexpression in yeast: a review, Yeast 8:423-488; van den Hondel, C. A. M.J. J. et al., 1991 Heterologous gene expression in filamentous fungi,in: More Gene Manipulations in Fungi, J. W. Bennet & L. L. Lasure, eds.,p. 396-428: Academic Press: San Diego; and van den Hondel, C. A. M. J.J. & Punt, P. J., 1991 Gene transfer systems and vector development forfilamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy, J.F. et al., eds., p. 1-28, Cambridge University Press: Cambridge), algae(Falciatore et al., 1999 Marine Biotechnology 1(3):239-251), ciliates ofthe types: Holotrichia, Peritrichia, Spirotrichia, Suctoria,Tetrahymena, Paramecium, Colpidium, Glaucoma, Platyophrya, Potomacus,Pseudocohnilembus, Euplotes, Engelmaniella, and Stylonychia, especiallyof the genus Stylonychia lemnae with vectors following a transformationmethod as described in WO 98/01572 and multicellular plant cells (seeSchmidt, R. and Willmitzer, L., 1988 High efficiency Agrobacteriumtumefaciens-mediated transformation of Arabidopsis thaliana leaf andcotyledon explants, Plant Cell Rep. 583-586); Plant Molecular Biologyand Biotechnology, C Press, Boca Raton, Fla., chapter 6/7, S.71-119(1993); F. F. White, B. Jenes et al., Techniques for Gene Transfer, in:Transgenic Plants, Vol. 1, Engineering and Utilization, eds. Kung und R.Wu, 12843, Academic Press: 1993; Potrykus, 1991 Annu. Rev. PlantPhysiol. Plant Molec. Biol. 42:205-225 and references cited therein) ormammalian cells. Suitable host cells are discussed further in Goeddel,Gene Expression Technology: Methods in Enzymology 185, Academic Press:San Diego, Calif. (1990). Alternatively, the recombinant expressionvector can be transcribed and translated in vitro, for example using T7promoter regulatory sequences and T7 polymerase.

[0086] Expression of proteins in prokaryotes is most often carried outwith vectors containing constitutive or inducible promoters directingthe expression of either fusion or non-fusion proteins. Fusion vectorsadd a number of amino acids to a protein encoded therein, usually to theamino terminus of the recombinant protein but also to the C-terminus orfused within suitable regions in the proteins. Such fusion vectorstypically serve three purposes: 1) to increase expression of arecombinant protein; 2) to increase the solubility of a recombinantprotein; and 3) to aid in the purification of a recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.

[0087] Typical fusion expression vectors include pGEX (Pharmacia BiotechInc; Smith, D. B. and Johnson, K. S., 1988 Gene 67:31-40), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.)which fuse glutathione S-transferase (GST), maltose E binding protein,or protein A, respectively, to the target recombinant protein. In oneembodiment, the coding sequence of the TFSRP is cloned into a pGEXexpression vector to create a vector encoding a fusion proteincomprising, from the N-terminus to the C-terminus, GST-thrombin cleavagesite-X protein. The fusion protein can be purified by affinitychromatography using glutathione-agarose resin. Recombinant TFSRPunfused to GST can be recovered by cleavage of the fusion protein withthrombin.

[0088] Examples of suitable inducible non-fusion E. coli expressionvectors include pTrc (Amann et al., 1988 Gene 69:301-315) and pET 11d(Studier et al., Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990) 60-89). Target gene expressionfrom the pTrc vector relies on host RNA polymerase transcription from ahybrid trp-lac fusion promoter. Target gene expression from the pET 11dvector relies on transcription from a T7 gn10-lac fusion promotermediated by a co-expressed viral RNA polymerase (T7 gn1). This viralpolymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from aresident λ prophage harboring a T7 gn1 gene under the transcriptionalcontrol of the lacUV 5 promoter.

[0089] One strategy to maximize recombinant protein expression is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the sequenceof the nucleic acid to be inserted into an expression vector so that theindividual codons for each amino acid are those preferentially utilizedin the bacterium chosen for expression, such as C. glutamicum (Wada etal., 1992 Nucleic Acids Res. 20:2111-2118). Such alteration of nucleicacid sequences of the invention can be carried out by standard DNAsynthesis techniques.

[0090] In another embodiment, the TFSRP expression vector is a yeastexpression vector. Examples of vectors for expression in yeast S.cerevisiae include pYepSec1 (Baldari, et al., 1987 Embo J. 6:229-234),pMFa (Kurjan and Herskowitz, 1982 Cell 30:933-943), pJRY88 (Schultz etal., 1987 Gene 54:113-123), and pYES2 (Invitrogen Corporation, SanDiego, Calif.). Vectors and methods for the construction of vectorsappropriate for use in other fungi, such as the filamentous fungi,include those detailed in: van den Hondel, C. A. M. J. J. & Punt, P. J.(1991) “Gene transfer systems and vector development for filamentousfungi, in: Applied Molecular Genetics of Fungi, J. F. Peberdy, et al.,eds., p. 1-28, Cambridge University Press: Cambridge.

[0091] Alternatively, the TFSRPs of the invention can be expressed ininsect cells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., 1983 Mol. Cell Biol.3:2156-2165) and the pVL series (Lucklow and Summers, 1989 Virology170:31-39).

[0092] In yet another embodiment, a TFSRP nucleic acid of the inventionis expressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, B., 1987Nature 329:840) and pMT2PC (Kaufman et al., 1987 EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2^(nd) , ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989.

[0093] In another embodiment, the recombinant mammalian expressionvector is capable of directing expression of the nucleic acidpreferentially in a particular cell type (e.g., tissue-specificregulatory elements are used to express the nucleic acid).Tissue-specific regulatory elements are known in the art. Non-limitingexamples of suitable tissue-specific promoters include the albuminpromoter (liver-specific; Pinkert et al., 1987 Genes Dev. 1:268-277),lymphoid-specific promoters (Calame and Eaton, 1988 Adv. Immunol.43:235-275), in particular promoters of T cell receptors (Winoto andBaltimore, 1989 EMBO J. 8:729-733) and immunoglobulins (Banerji et al.,1983 Cell 33:729-740; Queen and Baltimore, 1983 Cell 33:741-748),neuron-specific promoters (e.g., the neurofilament promoter; Byrne andRuddle, 1989 PNAS 86:5473-5477), pancreas-specific promoters (Edlund etal., 1985 Science 230:912-916), and mammary gland-specific promoters(e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and EuropeanApplication Publication No. 264,166). Developmentally-regulatedpromoters are also encompassed, for example, the murine hox promoters(Kessel and Gruss, 1990 Science 249:374-379) and the fetoproteinpromoter (Campes and Tilghman, 1989 Genes Dev. 3:537-546).

[0094] In another embodiment, the TFSRPs of the invention may beexpressed in unicellular plant cells (such as algae) (see Falciatore etal., 1999 Marine Biotechnology 1(3):239-251 and references therein) andplant cells from higher plants (e.g., the spermatophytes, such as cropplants). Examples of plant expression vectors include those detailed in:Becker, D., Kemper, E., Schell, J. and Masterson, R., 1992 New plantbinary vectors with selectable markers located proximal to the leftborder, Plant Mol. Biol. 20: 1195-1197; and Bevan, M. W., 1984 BinaryAgrobacterium vectors for plant transformation, Nucl. Acid. Res.12:8711-8721; Vectors for Gene Transfer in Higher Plants; in: TransgenicPlants, Vol. 1, Engineering and Utilization, eds.: Kung and R. Wu,Academic Press, 1993, S. 15-38.

[0095] A plant expression cassette preferably contains regulatorysequences capable of driving gene expression in plant cells and operablylinked so that each sequence can fulfill its function, for example,termination of transcription by polyadenylation signals. Preferredpolyadenylation signals are those originating from Agrobacteriumtumefaciens t-DNA such as the gene 3 known as octopine synthase of theT1-plasmid pTiACH5 (Gielen et al., 1984 EMBO J. 3:835) or functionalequivalents thereof but also all other terminators functionally activein plants are suitable.

[0096] As plant gene expression is very often not limited ontranscriptional levels, a plant expression cassette preferably containsother operably linked sequences like translational enhancers such as theoverdrive-sequence containing the 5′-untranslated leader sequence fromtobacco mosaic virus enhancing the protein per RNA ratio (Gallie et al.,1987 Nucl. Acids Research 15:8693-8711).

[0097] Plant gene expression has to be operably linked to an appropriatepromoter conferring gene expression in a timely, cell or tissue specificmanner. Preferred are promoters driving constitutive expression (Benfeyet al., 1989 EMBO J. 8:2195-2202) like those derived from plant viruseslike the 35S CAMV (Franck et al., 1980 Cell 21:285-294), the 19S CaMV(see also U.S. Pat. No. 5,352,605 and PCT Application No. WO 8402913) orplant promoters like those from Rubisco small subunit described in U.S.Pat. No. 4,962,028.

[0098] Other preferred sequences for use in plant gene expressioncassettes are targeting-sequences necessary to direct the gene productin its appropriate cell compartment (for review see Kermode, 1996 Crit.Rev. Plant Sci. 15(4):285-423 and references cited therein) such as thevacuole, the nucleus, all types of plastids like amyloplasts,chloroplasts, chromoplasts, the extracellular space, mitochondria, theendoplasmic reticulum, oil bodies, peroxisomes and other compartments ofplant cells.

[0099] Plant gene expression can also be facilitated via an induciblepromoter (for review see Gatz, 1997 Annu. Rev. Plant Physiol. Plant Mol.Biol. 48:89-108). Chemically inducible promoters are especially suitableif gene expression is wanted to occur in a time specific manner.Examples of such promoters are a salicylic acid inducible promoter (PCTApplication No. WO 95/19443), a tetracycline inducible promoter (Gatz etal., 1992 Plant J. 2:397-404) and an ethanol inducible promoter (PCTApplication No. WO 93/21334).

[0100] Also, suitable promoters responding to biotic or abiotic stressconditions are those such as the pathogen inducible PRP1-gene promoter(Ward et al., 1993 Plant. Mol. Biol. 22:361-366), the heat induciblehsp80-promoter from tomato (U.S. Pat. No. 5,187,267), cold induciblealpha-amylase promoter from potato (PCT Application No. WO 96/12814) orthe wound-inducible pinII-promoter (European Patent No. 375091). Forother examples of drought, cold, and salt-inducible promoters, such asthe RD29A promoter, see Yamaguchi-Shinozalei et al. (1993 Mol. Gen.Genet. 236:331-340).

[0101] Especially preferred are those promoters that confer geneexpression in specific tissues and organs, such as guard cells and theroot hair cells. Suitable promoters include the napin-gene promoter fromrapeseed (U.S. Pat. No. 5,608,152), the USP-promoter from Vicia faba(Baeumlein et al., 1991 Mol Gen Genet. 225(3):459-67), theoleosin-promoter from Arabidopsis (PCT Application No. WO 98/45461), thephaseolin-promoter from Phaseolus vulgaris (U.S. Pat. No. 5,504,200),the Bce4-promoter from Brassica (PCT Application No. WO 91/13980) or thelegumin B4 promoter (LeB4; Baeumlein et al., 1992 Plant Journal,2(2):233-9) as well as promoters conferring seed specific expression inmonocot plants like maize, barley, wheat, rye, rice, etc. Suitablepromoters to note are the lpt2 or lpt1-gene promoter from barley (PCTApplication No. WO 95/15389 and PCT Application No. WO 95/23230) orthose described in PCT Application No. WO 99/16890 (promoters from thebarley hordein-gene, rice glutelin gene, rice oryzin gene, rice prolamingene, wheat gliadin gene, wheat glutelin gene, maize zein gene, oatglutelin gene, Sorghum kasirin-gene and rye secalin gene).

[0102] Also especially suited are promoters that confer plastid-specificgene expression since plastids are the compartment where lipidbiosynthesis occurs. Suitable promoters are the viral RNA-polymerasepromoter described in PCT Application No. WO 95/16783 and PCTApplication No. WO 97/06250 and the clpP-promoter from Arabidopsisdescribed in PCT Application No. WO 99/46394.

[0103] The invention further provides a recombinant expression vectorcomprising a TFSRP DNA molecule of the invention cloned into theexpression vector in an antisense orientation. That is, the DNA moleculeis operatively linked to a regulatory sequence in a manner that allowsfor expression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to a TFSRP mRNA. Regulatory sequences operativelylinked to a nucleic acid molecule cloned in the antisense orientationcan be chosen which direct the continuous expression of the antisenseRNA molecule in a variety of cell types. For instance, viral promotersand/or enhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus wherein antisensenucleic acids are produced under the control of a high efficiencyregulatory region. The activity of the regulatory region can bedetermined by the cell type into which the vector is introduced. For adiscussion of the regulation of gene expression using antisense genessee Weintraub, H. et al., Antisense RNA as a molecular tool for geneticanalysis, Reviews—Trends in Genetics, Vol. 1(1) 1986 and Mol et al.,1990 FEBS Letters 268:427-430.

[0104] Another aspect of the invention pertains to host cells into whicha recombinant expression vector of the invention has been introduced.The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but they also apply to the progeny orpotential progeny of such a cell. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

[0105] A host cell can be any prokaryotic or eukaryotic cell. Forexample, a TFSRP can be expressed in bacterial cells such as C.glutamicum, insect cells, fungal cells or mammalian cells (such asChinese hamster ovary cells (CHO) or COS cells), algae, ciliates, plantcells, fungi or other microorganisms like C. glutamicum. Other suitablehost cells are known to those skilled in the art.

[0106] Vector DNA can be introduced into prokaryotic or eukaryotic cellsvia conventional transformation or transfection techniques. As usedherein, the terms “transformation”, “transfection”, “conjugation” and“transduction” are intended to refer to a variety of art-recognizedtechniques for introducing foreign nucleic acid (e.g., DNA) into a hostcell, including calcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection, natural competence,chemical-mediated transfer and electroporation. Suitable methods fortransforming or transfecting host cells including plant cells can befound in Sambrook, et al. (Molecular Cloning: A Laboratory Manual.2^(nd), ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989) and other laboratorymanuals such as Methods in Molecular Biology, 1995, Vol. 44,Agrobacterium protocols, ed: Gartland and Davey, Humana Press, Totowa,N.J. As biotic and abiotic stress tolerance is a general trait wished tobe inherited into a wide variety of plants like maize, wheat, rye, oat,triticale, rice, barley, soybean, peanut, cotton, rapeseed and canola,manihot, pepper, sunflower and tagetes, solanaceous plants like potato,tobacco, eggplant, and tomato, Vicia species, pea, alfalfa, bushy plants(coffee, cacao, tea), Salix species, trees (oil palm, coconut),perennial grasses and forage crops, these crop plants are also preferredtarget plants for a genetic engineering as one further embodiment of thepresent invention.

[0107] In particular, the invention provides a method of producing atransgenic plant with a TFSRP coding nucleic acid, wherein expression ofthe nucleic acid(s) in the plant results in increased tolerance toenvironmental stress as compared to a wild type variety of the plantcomprising: (a) transforming a plant cell with an expression vectorcomprising a TFSRP nucleic acid, and (b) generating from the plant cella transgenic plant with a increased tolerance to environmental stress ascompared to a wild type variety of the plant. The invention alsoprovides a method of increasing expression of a gene of interest withina host cell as compared to a wild type variety of the host cell, whereinthe gene of interest is transcribed in response to a TFSRP, comprising:(a) transforming the host cell with an expression vector comprising aTFSRP coding nucleic acid, and (b) expressing the TFSRP within the hostcell, thereby increasing the expression of the gene transcribed inresponse to the TFSRP, as compared to a wild type variety of the hostcell.

[0108] For such plant transformation, binary vectors such as pBinAR canbe used (Höfgen and Willmitzer, 1990 Plant Science 66:221-230).Construction of the binary vectors can be performed by ligation of thecDNA in sense or antisense orientation into the T-DNA. 5-prime to thecDNA a plant promoter activates transcription of the cDNA. Apolyadenylation sequence is located 3-prime to the cDNA. Tissue-specificexpression can be achieved by using a tissue specific promoter. Forexample, seed-specific expression can be achieved by cloning the napinor LeB4 or USP promoter 5-prime to the cDNA. Also, any other seedspecific promoter element can be used. For constitutive expressionwithin the whole plant, the CaMV 35S promoter can be used. The expressedprotein can be targeted to a cellular compartment using a signalpeptide, for example for plastids, mitochondria or endoplasmic reticulum(Kermode, 1996 Crit. Rev. Plant Sci. 4 (15):285-423). The signal peptideis cloned 5-prime in frame to the cDNA to archive subcellularlocalization of the fusion protein. Additionally, promoters that areresponsive to abiotic stresses can be used with, such as the Arabidopsispromoter RD29A, the nucleic acid sequences disclosed herein. One skilledin the art will recognize that the promoter used should be operativelylinked to the nucleic acid such that the promoter causes transcriptionof the nucleic acid which results in the synthesis of a mRNA whichencodes a polypeptide. Alternatively, the RNA can be an antisense RNAfor use in affecting subsequent expression of the same or another geneor genes.

[0109] Alternate methods of transfection include the direct transfer ofDNA into developing flowers via electroporation or Agrobacteriummediated gene transfer. Agrobacterium mediated plant transformation canbe performed using for example the GV3101(pMP90) (Koncz and Schell, 1986Mol. Gen. Genet. 204:383-396) or LBA4404 (Clontech) Agrobacteriumtumefaciens strain. Transformation can be performed by standardtransformation and regeneration techniques (Deblaere et al., 1994 Nucl.Acids. Res. 13:4777-4788; Gelvin, Stanton B. and Schilperoort, Robert A,Plant Molecular Biology Manual, 2^(nd) Ed.—Dordrecht: Kluwer AcademicPubl., 1995.—in Sect., Ringbuc Zentrale Signatur: BT11-P ISBN0-7923-2731-4; Glick, Bernard R.; Thompson, John E., Methods in PlantMolecular Biology and Biotechnology, Boca Raton: CRC Press, 1993.—360S., ISBN 0-8493-5164-2). For example, rapeseed can be transformed viacotyledon or hypocotyl transformation (Moloney et al., 1989 Plant cellReport 8:238-242; De Block et al., 1989 Plant Physiol. 91:694-701). Useof antibiotica for Agrobacterium and plant selection depends on thebinary vector and the Agrobacterium strain used for transformation.Rapeseed selection is normally performed using kanamycin as selectableplant marker. Agrobacterium mediated gene transfer to flax can beperformed using, for example, a technique described by Mlynarova et al.,1994 Plant Cell Report 13:282-285. Additionally, transformation ofsoybean can be performed using for example a technique described inEuropean Patent No. 0424 047, U.S. Pat. No. 5,322,783, European PatentNo. 0397 687, U.S. Pat. No. 5,376,543 or U.S. Pat. No. 5,169,770.Transformation of maize can be achieved by particle bombardment,polyethylene glycol mediated DNA uptake or via the silicon carbide fibertechnique. (See, for example, Freeling and Walbot “The maize handbook”Springer Verlag: New York (1993) ISBN 3-540-97826-7). A specific exampleof maize transformation is found in U.S. Pat. No. 5,990,387 and aspecific example of wheat transformation can be found in PCT ApplicationNo. WO 93/07256.

[0110] For stable transfection of mammalian cells, it is known that,depending upon the expression vector and transfection technique used,only a small fraction of cells may integrate the foreign DNA into theirgenome. In order to identify and select these integrants, a gene thatencodes a selectable marker (e.g., resistance to antibiotics) isgenerally introduced into the host cells along with the gene ofinterest. Preferred selectable markers include those which conferresistance to drugs, such as G418, hygromycin and methotrexate or inplants that confer resistance towards a herbicide such as glyphosate orglufosinate. Nucleic acid molecules encoding a selectable marker can beintroduced into a host cell on the same vector as that encoding a TFSRPor can be introduced on a separate vector. Cells stably transfected withthe introduced nucleic acid molecule can be identified by, for example,drug selection (e.g., cells that have incorporated the selectable markergene will survive, while the other cells die).

[0111] To create a homologous recombinant microorganism, a vector isprepared which contains at least a portion of a TFSRP gene into which adeletion, addition or substitution has been introduced to thereby alter,e.g., functionally disrupt, the TFSRP gene. Preferably, the TFSRP geneis a Physcomitrella patens TFSRP gene, but it can be a homolog from arelated plant or even from a mammalian, yeast, or insect source. In apreferred embodiment, the vector is designed such that, upon homologousrecombination, the endogenous TFSRP gene is functionally disrupted(i.e., no longer encodes a functional protein; also referred to as aknock-out vector). Alternatively, the vector can be designed such that,upon homologous recombination, the endogenous TFSRP gene is mutated orotherwise altered but still encodes a functional protein (e.g., theupstream regulatory region can be altered to thereby alter theexpression of the endogenous TFSRP). To create a point mutation viahomologous recombination, DNA-RNA hybrids can be used in a techniqueknown as chimeraplasty (Cole-Strauss et al., 1999 Nucleic Acids Research27(5):1323-1330 and Kmiec, 1999 Gene therapy American Scientist.87(3):240-247). Homologous recombination procedures in Physcomitrellapatens are also well known in the art and are contemplated for useherein.

[0112] Whereas in the homologous recombination vector, the alteredportion of the TFSRP gene is flanked at its 5′ and 3′ ends by anadditional nucleic acid molecule of the TFSRP gene to allow forhomologous recombination to occur between the exogenous TFSRP genecarried by the vector and an endogenous TFSRP gene, in a microorganismor plant. The additional flanking TFSRP nucleic acid molecule is ofsufficient length for successful homologous recombination with theendogenous gene. Typically, several hundreds of base pairs up tokilobases of flanking DNA (both at the 5′ and 3′ ends) are included inthe vector (see e.g., Thomas, K. R., and Capecchi, M. R., 1987 Cell51:503 for a description of homologous recombination vectors or Streppet al., 1998 PNAS, 95 (8):4368-4373 for cDNA based recombination inPhyscomitrella patens). The vector is introduced into a microorganism orplant cell (e.g., via polyethylene glycol mediated DNA), and cells inwhich the introduced TFSRP gene has homologously recombined with theendogenous TFSRP gene are selected using art-known techniques.

[0113] In another embodiment, recombinant microorganisms can be producedthat contain selected systems which allow for regulated expression ofthe introduced gene. For example, inclusion of a TFSRP gene on a vectorplacing it under control of the lac operon permits expression of theTFSRP gene only in the presence of IPTG. Such regulatory systems arewell known in the art.

[0114] A host cell of the invention, such as a prokaryotic or eukaryotichost cell in culture, can be used to produce (i.e., express) a TFSRP.Accordingly, the invention further provides methods for producing TFSRPsusing the host cells of the invention. In one embodiment, the methodcomprises culturing the host cell of invention (into which a recombinantexpression vector encoding a TFSRP has been introduced, or into whichgenome has been introduced a gene encoding a wild-type or altered TFSRP)in a suitable medium until TFSRP is produced. In another embodiment, themethod further comprises isolating TFSRPs from the medium or the hostcell.

[0115] Another aspect of the invention pertains to isolated TFSRPs, andbiologically active portions thereof. An “isolated” or “purified”protein or biologically active portion thereof is free of some of thecellular material when produced by recombinant DNA techniques, orchemical precursors or other chemicals when chemically synthesized. Thelanguage “substantially free of cellular material” includes preparationsof TFSRP in which the protein is separated from some of the cellularcomponents of the cells in which it is naturally or recombinantlyproduced. In one embodiment, the language “substantially free ofcellular material” includes preparations of a TFSRP having less thanabout 30% (by dry weight) of non-TFSRP material (also referred to hereinas a “contaminating protein”), more preferably less than about 20% ofnon-TFSRP material, still more preferably less than about 10% ofnon-TFSRP material, and most preferably less than about 5% non-TFSRPmaterial.

[0116] When the TFSRP or biologically active portion thereof isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the protein preparation. The language “substantiallyfree of chemical precursors or other chemicals” includes preparations ofTFSRP in which the protein is separated from chemical precursors orother chemicals that are involved in the synthesis of the protein. Inone embodiment, the language “substantially free of chemical precursorsor other chemicals” includes preparations of a TFSRP having less thanabout 30% (by dry weight) of chemical precursors or non-TFSRP chemicals,more preferably less than about 20% chemical precursors or non-TFSRPchemicals, still more preferably less than about 10% chemical precursorsor non-TFSRP chemicals, and most preferably less than about 5% chemicalprecursors or non-TFSRP chemicals. In preferred embodiments, isolatedproteins, or biologically active portions thereof, lack contaminatingproteins from the same organism from which the TFSRP is derived.Typically, such proteins are produced by recombinant expression of, forexample, a Physcomitrella patens TFSRP in plants other thanPhyscomitrella patens or microorganisms such as C. glutamicum, ciliates,algae or fungi.

[0117] The nucleic acid molecules, proteins, protein homologs, fusionproteins, primers, vectors, and host cells described herein can be usedin one or more of the following methods: identification ofPhyscomitrella patens and related organisms; mapping of genomes oforganisms related to Physcomitrella patens; identification andlocalization of Physcomitrella patens sequences of interest;evolutionary studies; determination of TFSRP regions required forfunction; modulation of a TFSRP activity; modulation of the metabolismof one or more cell functions; modulation of the transmembrane transportof one or more compounds; and modulation of stress resistance.

[0118] The moss Physcomitrella patens represents one member of themosses. It is related to other mosses such as Ceratodon purpureus whichis capable of growth in the absence of light. Mosses like Ceratodon andPhyscomitrella share a high degree of homology on the DNA sequence andpolypeptide level allowing the use of heterologous screening of DNAmolecules with probes evolving from other mosses or organisms, thusenabling the derivation of a consensus sequence suitable forheterologous screening or functional annotation and prediction of genefunctions in third species. The ability to identify such functions cantherefore have significant relevance, e.g., prediction of substratespecificity of enzymes. Further, these nucleic acid molecules may serveas reference points for the mapping of moss genomes, or of genomes ofrelated organisms.

[0119] The TFSRP nucleic acid molecules of the invention have a varietyof uses. Most importantly, the nucleic acid and amino acid sequences ofthe present invention can be used to transform plants, thereby inducingtolerance to stresses such as drought, high salinity and cold. Thepresent invention therefore provides a transgenic plant transformed by aTFSRP nucleic acid, wherein expression of the nucleic acid sequence inthe plant results in increased tolerance to environmental stress ascompared to a wild type variety of the plant. The transgenic plant canbe a monocot or a dicot. The invention further provides that thetransgenic plant can be selected from maize, wheat, rye, oat, triticale,rice, barley, soybean, peanut, cotton, rapeseed, canola, manihot,pepper, sunflower, tagetes, solanaceous plants, potato, tobacco,eggplant, tomato, Vicia species, pea, alfalfa, coffee, cacao, tea, Salixspecies, oil palm, coconut, perennial grass and forage crops, forexample.

[0120] In particular, the present invention describes using theexpression of APS-2, ZF-2, ZF-3, ZF-4, ZF-5, MYB-1, CABF-3 and SFL-1 ofPhyscomitrella patens to engineer drought-tolerant, salt-tolerant and/orcold-tolerant plants. This strategy has herein been demonstrated forArabidopsis thaliana, Rapeseed/Canola, soybeans, corn and wheat but itsapplication is not restricted to these plants. Accordingly, theinvention provides a transgenic plant containing a TFSRP selected fromAPS-2 (SEQ ID NO:17), ZF-2 (SEQ ID NO:18), ZF-3 (SEQ ID NO:19), ZF-4(SEQ ID NO:20), ZF-5 (SEQ ID NO:21), MYB-1 (SEQ ID NO:22), CABF-3 (SEQID NO:23) and SFL-1 (SEQ ID NO:24), wherein the environmental stress isdrought, increased salt or decreased or increased temperature. Inpreferred embodiments, the environmental stress is drought or decreasedtemperature

[0121] The present invention also provides methods of modifying stresstolerance of a plant comprising, modifying the expression of a TFSRP inthe plant. The invention provides that this method can be performed suchthat the stress tolerance is either increased or decreased. Inparticular, the present invention provides methods of producing atransgenic plant having an increased tolerance to environmental stressas compared to a wild type variety of the plant comprising increasingexpression of a TFSRP in a plant.

[0122] The methods of increasing expression of TFSRPs can be usedwherein the plant is either transgenic or not transgenic. In cases whenthe plant is transgenic, the plant can be transformed with a vectorcontaining any of the above described TFSRP coding nucleic acids, or theplant can be transformed with a promoter that directs expression ofnative TFSRP in the plant, for example. The invention provides that sucha promoter can be tissue specific. Furthermore, such a promoter can bedevelopmentally regulated. Alternatively, non-transgenic plants can havenative TFSRP expression modified by inducing a native promoter.

[0123] The expression of APS-2 (SEQ ID NO:17), ZF-2 (SEQ ID NO:18), ZF-3(SEQ ID NO:19), ZF-4 (SEQ ID NO:20), ZF-5 (SEQ ID NO:21), MYB-1 (SEQ IDNO:22), CABF-3 (SEQ ID NO:23) or SFL-1 (SEQ ID NO:24) in target plantscan be accomplished by, but is not limited to, one of the followingexamples: (a) constitutive promoter, (b) stress-inducible promoter, (c)chemical-induced promoter, and (d) engineered promoter over-expressionwith for example zinc-finger derived transcription factors (Greisman andPabo, 1997 Science 275:657). The later case involves identification ofthe APS-2 (SEQ ID NO:17), ZF-2 (SEQ ID NO:18), ZF-3 (SEQ ID NO:19), ZF-4(SEQ ID NO:20), ZF-5 (SEQ ID NO:21), MYB-1 (SEQ ID NO:22), CABF-3 (SEQID NO:23) or SFL-1 (SEQ ID NO:24) homologs in the target plant as wellas from its promoter. Zinc-finger-containing recombinant transcriptionfactors are engineered to specifically interact with the APS-2 (SEQ IDNO:17), ZF-2 (SEQ ID NO:18), ZF-3 (SEQ ID NO:19), ZF-4 (SEQ ID NO:20),ZF-5 (SEQ ID NO:21), MYB-1 (SEQ ID NO:22), CABF-3 (SEQ ID NO:23) orSFL-1 (SEQ ID NO:24) homolog and transcription of the corresponding geneis activated.

[0124] In addition to introducing the TFSRP nucleic acid sequences intotransgenic plants, these sequences can also be used to identify anorganism as being Physcomitrella patens or a close relative thereof.Also, they may be used to identify the presence of Physcomitrella patensor a relative thereof in a mixed population of microorganisms. Theinvention provides the nucleic acid sequences of a number ofPhyscomitrella patens genes; by probing the extracted genomic DNA of aculture of a unique or mixed population of microorganisms understringent conditions with a probe spanning a region of a Physcomitrellapatens gene which is unique to this organism, one can ascertain whetherthis organism is present.

[0125] Further, the nucleic acid and protein molecules of the inventionmay serve as markers for specific regions of the genome. This hasutility not only in the mapping of the genome, but also in functionalstudies of Physcomitrella patens proteins. For example, to identify theregion of the genome to which a particular Physcomitrella patensDNA-binding protein binds, the Physcomitrella patens genome could bedigested, and the fragments incubated with the DNA-binding protein.Those fragments that bind the protein may be additionally probed withthe nucleic acid molecules of the invention, preferably with readilydetectable labels. Binding of such a nucleic acid molecule to the genomefragment enables the localization of the fragment to the genome map ofPhyscomitrella patens, and, when performed multiple times with differentenzymes, facilitates a rapid determination of the nucleic acid sequenceto which the protein binds. Further, the nucleic acid molecules of theinvention may be sufficiently homologous to the sequences of relatedspecies such that these nucleic acid molecules may serve as markers forthe construction of a genomic map in related mosses.

[0126] The TFSRP nucleic acid molecules of the invention are also usefulfor evolutionary and protein structural studies. The metabolic andtransport processes in which the molecules of the invention participateare utilized by a wide variety of prokaryotic and eukaryotic cells; bycomparing the sequences of the nucleic acid molecules of the presentinvention to those encoding similar enzymes from other organisms, theevolutionary relatedness of the organisms can be assessed. Similarly,such a comparison permits an assessment of which regions of the sequenceare conserved and which are not, which may aid in determining thoseregions of the protein that are essential for the functioning of theenzyme. This type of determination is of value for protein engineeringstudies and may give an indication of what the protein can tolerate interms of mutagenesis without losing function.

[0127] Manipulation of the TFSRP nucleic acid molecules of the inventionmay result in the production of TFSRPs having functional differencesfrom the wild-type TFSRPs. These proteins may be improved in efficiencyor activity, may be present in greater numbers in the cell than isusual, or may be decreased in efficiency or activity.

[0128] There are a number of mechanisms by which the alteration of aTFSRP of the invention may directly affect stress response and/or stresstolerance. In the case of plants expressing TFSRPs, increased transportcan lead to improved salt and/or solute partitioning within the planttissue and organs. By either increasing the number or the activity oftransporter molecules which export ionic molecules from the cell, it maybe possible to affect the salt tolerance of the cell.

[0129] The effect of the genetic modification in plants, C. glutamicum,fungi, algae, or ciliates on stress tolerance can be assessed by growingthe modified microorganism or plant under less than suitable conditionsand then analyzing the growth characteristics and/or metabolism of theplant. Such analysis techniques are well known to one skilled in theart, and include dry weight, wet weight, protein synthesis, carbohydratesynthesis, lipid synthesis, evapotranspiration rates, general plantand/or crop yield, flowering, reproduction, seed setting, root growth,respiration rates, photosynthesis rates, etc. (Applications of HPLC inBiochemistry in: Laboratory Techniques in Biochemistry and MolecularBiology, vol. 17; Rehm et al., 1993 Biotechnology, vol. 3, Chapter III:Product recovery and purification, page 469-714, VCH: Weinheim; Belter,P. A. et al., 1988 Bioseparations: downstream processing forbiotechnology, John Wiley and Sons; Kennedy, J. F. and Cabral, J. M. S.,1992 Recovery processes for biological materials, John Wiley and Sons;Shaeiwitz, J. A. and Henry, J. D., 1988 Biochemical separations, in:Ulmann's Encyclopedia of Industrial Chemistry, vol. B3, Chapter 11, page1-27, VCH: Weinheim; and Dechow, F. J. (1989) Separation andpurification techniques in biotechnology, Noyes Publications).

[0130] For example, yeast expression vectors comprising the nucleicacids disclosed herein, or fragments thereof, can be constructed andtransformed into Saccharomyces cerevisiae using standard protocols. Theresulting transgenic cells can then be assayed for fail or alteration oftheir tolerance to drought, salt, and temperature stress. Similarly,plant expression vectors comprising the nucleic acids disclosed herein,or fragments thereof, can be constructed and transformed into anappropriate plant cell such as Arabidopsis, soy, rape, maize, wheat,Medicago truncatula, etc., using standard protocols. The resultingtransgenic cells and/or plants derived there from can then be assayedfor fail or alteration of their tolerance to drought, salt, andtemperature stress.

[0131] The engineering of one or more TFSRP genes of the invention mayalso result in TFSRPs having altered activities which indirectly impactthe stress response and/or stress tolerance of algae, plants, ciliatesor fungi or other microorganisms like C. glutamicum. For example, thenormal biochemical processes of metabolism result in the production of avariety of products (e.g., hydrogen peroxide and other reactive oxygenspecies) which may actively interfere with these same metabolicprocesses (for example, peroxynitrite is known to nitrate tyrosine sidechains, thereby inactivating some enzymes having tyrosine in the activesite (Groves, J. T., 1999 Curr. Opin. Chem. Biol. 3(2):226-235). Whilethese products are typically excreted, cells can be genetically alteredto transport more products than is typical for a wild-type cell. Byoptimizing the activity of one or more TFSRPs of the invention which areinvolved in the export of specific molecules, such as salt molecules, itmay be possible to improve the stress tolerance of the cell.

[0132] Additionally, the sequences disclosed herein, or fragmentsthereof, can be used to generate knockout mutations in the genomes ofvarious organisms, such as bacteria, mammalian cells, yeast cells, andplant cells (Girke, T., 1998 The Plant Journal 15:3948). The resultantknockout cells can then be evaluated for their ability or capacity totolerate various stress conditions, their response to various stressconditions, and the effect on the phenotype and/or genotype of themutation. For other methods of gene inactivation see U.S. Pat. No.6,004,804 “Non-Chimeric Mutational Vectors” and Puttaraju et al., 1999Spliceosome-mediated RNA trans-splicing as a tool for gene therapyNature Biotechnology 17:246-252.

[0133] The aforementioned mutagenesis strategies for TFSRPs resulting inincreased stress resistance are not meant to be limiting; variations onthese strategies will be readily apparent to one skilled in the art.Using such strategies, and incorporating the mechanisms disclosedherein, the nucleic acid and protein molecules of the invention may beutilized to generate algae, ciliates, plants, fungi or othermicroorganisms like C. glutamicum expressing mutated TFSRP nucleic acidand protein molecules such that the stress tolerance is improved.

[0134] The present invention also provides antibodies that specificallybind to a TFSRP, or a portion thereof, as encoded by a nucleic aciddescribed herein. Antibodies can be made by many well-known methods(See, e.g. Harlow and Lane, “Antibodies; A Laboratory Manual” ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., (1988)). Briefly,purified antigen can be injected into an animal in an amount and inintervals sufficient to elicit an immune response. Antibodies can eitherbe purified directly, or spleen cells can be obtained from the animal.The cells can then fused with an immortal cell line and screened forantibody secretion. The antibodies can be used to screen nucleic acidclone libraries for cells secreting the antigen. Those positive clonescan then be sequenced. (See, for example, Kelly et al., 1992Bio/Technology 10:163-167; Bebbington et al., 1992 Bio/Technology10:169-175).

[0135] The phrases “selectively binds” and “specifically binds” with thepolypeptide refer to a binding reaction that is determinative of thepresence of the protein in a heterogeneous population of proteins andother biologics. Thus, under designated immunoassay conditions, thespecified antibodies bound to a particular protein do not bind in asignificant amount to other proteins present in the sample. Selectivebinding of an antibody under such conditions may require an antibodythat is selected for its specificity for a particular protein. A varietyof immunoassay formats may be used to select antibodies that selectivelybind with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select antibodies selectivelyimmunoreactive with a protein. See Harlow and Lane “Antibodies, ALaboratory Manual” Cold Spring Harbor Publications, New York, (1988),for a description of immunoassay formats and conditions that could beused to determine selective binding.

[0136] In some instances, it is desirable to prepare monoclonalantibodies from various hosts. A description of techniques for preparingsuch monoclonal antibodies may be found in Stites et al., editors,“Basic and Clinical Immunology,” (Lange Medical Publications, Los Altos,Calif., Fourth Edition) and references cited therein, and in Harlow andLane (“Antibodies, A Laboratory Manual” Cold Spring Harbor Publications,New York, 1988).

[0137] Throughout this application, various publications are referenced.The disclosures of all of these publications and those references citedwithin those publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention pertains.

[0138] It should also be understood that the foregoing relates topreferred embodiments of the present invention and that numerous changesmay be made therein without departing from the scope of the invention.The invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof, which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims.

EXAMPLES Example 1

[0139] Growth of Physcomitrella patens Cultures

[0140] For this study, plants of the species Physcomitrella patens(Hedw.) B.S.G. from the collection of the genetic studies section of theUniversity of Hamburg were used. They originate from the strain 16/14collected by H. L. K. Whitehouse in Gransden Wood, Huntingdonshire(England), which was subcultured from a spore by Engel (1968, Am. J.Bot. 55, 438-446). Proliferation of the plants was carried out by meansof spores and by means of regeneration of the gametophytes. Theprotonema developed from the haploid spore as a chloroplast-richchloronema and chloroplast-low caulonema, on which buds formed afterapproximately 12 days. These grew to give gametophores bearingantheridia and archegonia. After fertilization, the diploid sporophytewith a short seta and the spore capsule resulted, in which themeiospores matured.

[0141] Culturing was carried out in a climatic chamber at an airtemperature of 25° C. and light intensity of 55 micromols^(−1m2) (whitelight; Philips TL 65W/25 fluorescent tube) and a light/dark change of16/8 hours. The moss was either modified in liquid culture using Knopmedium according to Reski and Abel (1985, Planta 165:354-358) orcultured on Knop solid medium using 1% oxoid agar (Unipath, Basingstoke,England). The protonemas used for RNA and DNA isolation were cultured inaerated liquid cultures. The protonemas were comminuted every 9 days andtransferred to fresh culture medium.

Example 2

[0142] Total DNA Isolation from Plants

[0143] The details for the isolation of total DNA relate to the workingup of one gram fresh weight of plant material. The materials usedinclude the following buffers: CTAB buffer: 2% (w/v)N-cethyl-N,N,N-trimethylammonium bromide (CTAB); 100 mM Tris HCl pH 8.0;1.4 M NaCl; 20 mM EDTA; N-Laurylsarcosine buffer: 10% (w/v)N-laurylsarcosine; 100 mM Tris HCl pH 8.0; 20 mM EDTA.

[0144] The plant material was triturated under liquid nitrogen in amortar to give a fine powder and transferred to 2 ml Eppendorf vessels.The frozen plant material was then covered with a layer of 1 ml ofdecomposition buffer (1 ml CTAB buffer, 100 μl of N-laurylsarcosinebuffer, 20 μl of β-mercaptoethanol and 10 μl of proteinase K solution,10 mg/ml) and incubated at 60° C. for one hour with continuous shaking.The homogenate obtained was distributed into two Eppendorf vessels (2ml) and extracted twice by shaking with the same volume ofchloroform/isoamyl alcohol (24:1). For phase separation, centrifugationwas carried out at 8000×g and room temperature for 15 minutes in eachcase. The DNA was then precipitated at −70° C. for 30 minutes usingice-cold isopropanol. The precipitated DNA was sedimented at 4° C. and10,000 g for 30 minutes and resuspended in 180 μl of TE buffer (Sambrooket al., 1989, Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6).For further purification, the DNA was treated with NaCl (1.2 M finalconcentration) and precipitated again at −70° C. for 30 minutes usingtwice the volume of absolute ethanol. After a washing step with 70%ethanol, the DNA was dried and subsequently taken up in 50 μl ofH₂O+RNAse (50 mg/ml final concentration). The DNA was dissolvedovernight at 4° C. and the RNAse digestion was subsequently carried outat 37° C. for 1 hour. Storage of the DNA took place at 4° C.

Example 3

[0145] Isolation of Total RNA and Poly-(A)+ RNA and cDNA LibraryConstruction from Physcomitrella patens

[0146] For the investigation of transcripts, both total RNA andpoly-(A)⁺ RNA were isolated. The total RNA was obtained from wild-type 9day old protonemata following the GTC-method (Reski et al. 1994, Mol.Gen. Genet., 244:352-359). The Poly(A)+ RNA was isolated using DynaBeads^(R) (Dynal, Oslo, Norway) following the instructions of themanufacturers protocol. After determination of the concentration of theRNA or of the poly(A)+ RNA, the RNA was precipitated by addition of 1/10volumes of 3 M sodium acetate pH 4.6 and 2 volumes of ethanol and storedat −70° C.

[0147] For cDNA library construction, first strand synthesis wasachieved using Murine Leukemia Virus reverse transcriptase (Roche,Mannheim, Germany) and oligo-d(T)-primers, second strand synthesis byincubation with DNA polymerase I, Klenow enzyme and RNAseH digestion at12° C. (2 hours), 16° C. (1 hour) and 22° C. (1 hour). The reaction wasstopped by incubation at 65° C. (10 minutes) and subsequentlytransferred to ice. Double stranded DNA molecules were blunted byT4-DNA-polymerase (Roche, Mannheim) at 37° C. (30 minutes). Nucleotideswere removed by phenol/chloroform extraction and Sephadex G50 spincolumns. EcoRI adapters (Pharmacia, Freiburg, Germany) were ligated tothe cDNA ends by T4-DNA-ligase (Roche, 12° C., overnight) andphosphorylated by incubation with polynucleotide kinase (Roche, 37° C.,30 minutes). This mixture was subjected to separation on a low meltingagarose gel. DNA molecules larger than 300 base pairs were eluted fromthe gel, phenol extracted, concentrated on Elutip-D-columns (Schleicherand Schuell, Dassel, Germany) and were ligated to vector arms and packedinto lambda ZAPII phages or lambda ZAP-Express phages using the GigapackGold Kit (Stratagene, Amsterdam, Netherlands) using material andfollowing the instructions of the manufacturer.

Example 4

[0148] Sequencing and Function Annotation of Physcomitrella patens ESTs

[0149] cDNA libraries as described in Example 3 were used for DNAsequencing according to standard methods, and in particular, by thechain termination method using the ABI PRISM Big Dye Terminator CycleSequencing Ready Reaction Kit (Perkin-Elmer, Weiterstadt, Germany).Random Sequencing was carried out subsequent to preparative plasmidrecovery from cDNA libraries via in vivo mass excision,retransformation, and subsequent plating of DH10B on agar plates(material and protocol details from Stratagene, Amsterdam, Netherlands.Plasmid DNA was prepared from overnight grown E. coli cultures grown inLuria-Broth medium containing ampicillin (see Sambrook et al. 1989 ColdSpring Harbor Laboratory Press: ISBN 0-87969-309-6) on a Qiagene DNApreparation robot (Qiagen, Hilden) according to the manufacturer'sprotocols. Sequencing primers with the following nucleotide sequenceswere used: 5′-GAGGAAACAGCTATGAGC-3′ SEQ ID NO:255′-CTAAAGGGAACAAAAGCTG-3′ SEQ ID NO:26 5′-TGTAAAACGACGGCCAGT-3′ SEQ IDNO:27

[0150] Sequences were processed and annotated using the software packageEST-MAX commercially provided by Bio-Max (Munich, Germany). The programincorporates practically all bioinformatics methods important forfunctional and structural characterization of protein sequences. Forreference the website at pedant.mips.biochem.mpg.de. The most importantalgorithms incorporated in EST-MAX are: FASTA: Very sensitive sequencedatabase searches with estimates of statistical significance; Pearson W.R. (1990) Rapid and sensitive sequence comparison with FASTP and FASTA.Methods Enzymol. 183:63-98; BLAST: Very sensitive sequence databasesearches with estimates of statistical significance. Altschul S. F.,Gish W., Miller W., Myers E. W., and Lipman D. J. Basic local alignmentsearch tool. Journal of Molecular Biology 215:403-10; PREDATOR:High-accuracy secondary structure prediction from single and multiplesequences. Frishman, D. and Argos, P. (1997) 75% accuracy in proteinsecondary structure prediction. Proteins, 27:329-335; CLUSTAL W:Multiple sequence alignment. Thompson, J. D., Higgins, D. G. and Gibson,T. J. (1994) CLUSTAL W: improving the sensitivity of progressivemultiple sequence alignment through sequence weighting,positions-specific gap penalties and weight matrix choice. Nucleic AcidsResearch, 22:4673-4680; TMAP: Transmembrane region prediction frommultiply aligned sequences. Persson, B. and Argos, P. (1994) Predictionof transmembrane segments in proteins utilizing multiple sequencealignments. J. Mol. Biol. 237:182-192; ALOM2: Transmembrane regionprediction from single sequences. Klein, P., Kanehisa, M., and DeLisi,C. Prediction of protein function from sequence properties: Adiscriminate analysis of a database. Biochim. Biophys. Acta 787:221-226(1984). Version 2 by Dr. K. Nakai; PROSEARCH: Detection of PROSITEprotein sequence patterns. Kolakowski L. F. Jr., Leunissen J. A. M.,Smith J. E. (1992) ProSearch: fast searching of protein sequences withregular expression patterns related to protein structure and function.Biotechniques 13, 919-921; BLIMPS: Similarity searches against adatabase of ungapped blocks. J. C. Wallace and Henikoff S., (1992);PATMAT: A searching and extraction program for sequence, pattern andblock queries and databases, CABIOS 8:249-254. Written by Bill Alford.

Example 5

[0151] Identification of Physcomitrella patens ORFs Corresponding toAPS-2, ZF-2, ZF-3, ZF-4, ZF-5, MYB-1, CABF-3 and SFL-1

[0152] The Physcomitrella patens partial cDNAs (ESTs) shown in Table 1below were identified in the Physcomitrella patens EST sequencingprogram using the program EST-MAX through BLAST analysis. The SequenceIdentification Numbers corresponding to these ESTs are as follows: APS-2(SEQ ID NO:1), ZF-2 (SEQ ID NO:2), ZF-3 (SEQ ID NO:3), ZF-4 (SEQ IDNO:4), ZF-5 (SEQ ID NO:5), MYB-1 (SEQ ID NO:6), CABF-3 (SEQ ID NO:7) andSFL-1 (SEQ ID NO:8). TABLE 1 Functional ORF Name categories FunctionSequence code position PpAPS-2 CBF/Transcription AP2 domain containingc_pp001007077f 592-92 factor protein RAP2.11 PpZF-2 Transcription factorzinc finger protein c_pp004033187r 1688-765 PpZF-3 Transcription factorBRCA1-associated c_pp004042321r 1-500 RING domain protein PpZF-4Transcription factor zinc finger protein c_pp004059097r 701-1216 ZNF216PpZF-5 Transcription factor transcription factor-like c_pp004046041r1-675 protein PpMYB-1 Transcription factor transcription factors_pp002016030r 2-505 PpCABF-3 Transcription factor transcription factor,c_pp004040113r 221-535 CCAAT-binding, chain A PpSFL-1 Transcriptionfactor transcription initiation s_pp001105041r 598-158 factor sigma A

[0153] TABLE 2 Degree of amino acid identity and similarity of PpCBF-3and other homologous proteins (Pairwise comparison program was used: gappenalty: 10; gap extension penalty: 0.1; score matrix: blosum62)Swiss-Prot # O23310 P25209 Q9LFI3 O23633 Q9ZQC3 Protein name Ccaat-Ccaat- Transcription Transcription Putative binding binding factor nf-y,factor ccaat- transcription transcription ccaat- binding factor factorbinding-like transcription subunit a subunit a protein factor SpeciesArabidopsis Zea mays Arabidopsis Arabidopsis Arabidopsis thaliana(Maize) thaliana thaliana thaliana (Mouse-ear (Mouse-ear (Mouse-ear(Mouse-ear cress) cress) cress) cress) Identity % 53% 49% 42% 43% 62%Similarity % 58% 58% 53% 51% 66%

[0154] TABLE 3 Degree of amino acid identity and similarity of PpZF-2and other homologous proteins (Pairwise comparison program was used: gappenalty: 10; gap extension penalty: 0.1; score matrix: blosum62)Swiss-Prot # O24008 Q9LUR1 Q9XF63 Q9XF64 Q9LZJ6 Protein Zinc finger Ringzinc Ring-h2 zinc Ring-h2 Ring-h2 name protein finger finger proteinzinc finger zinc finger protein-like (atl3) protein atl5 protein atl5Species Arabidopsis Arabidopsis Arabidopsis Arabidopsis Arabidopsisthaliana thaliana thaliana thaliana thaliana (Mouse-ear (Mouse-ear(Mouse-ear (Mouse-ear (Mouse-ear cress) cress) cress) cress) cress)Identity % 27% 26% 25% 20% 19% Similarity % 35% 35% 34% 28% 28%

[0155] TABLE 4 Degree of amino acid identity and similarity of PpZF-3and other homologous proteins (Pairwise comparison program was used: gappenalty: 10; gap extension penalty: 0.1; score matrix: blosum62)Swiss-Prot # Q9SMX5 O04097 Q9UQR3 Q9XZQ1 Q9XZQ2 Protein name Gcn4-Brcal- Centaurin Centaurin Centaurin complementing associated beta2 beta1a beta 1b protein (gcp 1) ring domain proteinisolog Species ArabidopsisArabidopsis Homo Caenorhabditis Caenorhabditis thaliana thaliana sapienselegans elegans (Mouse-ear (Mouse-ear (Human) cress) cress) Identity %41% 37% 24% 21% 22% Similarity % 54% 49% 32% 31% 34%

[0156] TABLE 4 Degree of amino acid identity and similarity of PpZF-4and other homologous proteins (Pairwise comparison program was used: gappenalty: 10; gap extension penalty: 0.1; score matrix: blosum62)Swiss-Prot # Q9LXI5 O88878 O76080 Q9ZNU9 O96038 Protein name Zincfinger- Zinc finger Zinc finger Putative zinc Pem-6 like protein proteinprotein 216 finger protein znf216 Species Arabidopsis Mus HomoArabidopsis Ciona thaliana musculus sapiens thaliana savignyi (Mouse-ear(Mouse) (Human) (Mouse-ear cress) cress) Identity % 39% 34% 34% 35% 32%Similarity % 53% 45% 45% 50% 49%

[0157] TABLE 6 Degree of amino acid identity and similarity of PpZF-5and other homologous proteins (Pairwise comparison program was used: gappenalty: 10; gap extension penalty: 0.1; score matrix: blosum62)Swiss-Prot # Q9SZW1 Q9ZTR9 Q9SYQ6 Q9ZTX9 O23661 Protein nameTranscription Auxin Auxin Auxin Ettin protein factor-like responseresponse response protein factor 8 factor 7 factor 4 Species ArabidopsisArabidopsis Arabidopsis Arabidopsis Arabidopsis thaliana thalianathaliana thaliana thaliana (Mouse-ear (Mouse-ear (Mouse-ear (Mouse-earcress) (Mouse-ear cress) cress) cress) cress) Identity % 39% 23% 25% 25%25% Similarity % 50% 32% 33% 32% 35%

[0158] TABLE 7 Degree of amino acid identity and similarity of PpAPS-2and other homologous proteins (Pairwise comparison program was used: gappenalty: 10; gap extension penalty: 0.1; score matrix: blosum62)Swiss-Prot # Q9SJR0 O22174 O04682 Q9SW63 Q9SGJ6 Protein name Putativeap2 Putative ap2 Pathogenesis- Tiny-like Transcription domain domainrelated genes protein factor dreb 1a transcription containingtranscriptional factor protein activator pti6 Species ArabidopsisArabidopsis Lycopersicon Arabidopsis Arabidopsis thaliana thalianaesculentum thaliana thaliana (Mouse-ear (Mouse-ear (Tomato) (Mouse-ear(Mouse-ear cress) cress) cress) cress) Identity % 18% 19% 15% 15% 16%Similarity % 23% 29% 20% 25% 24%

[0159] TABLE 8 Degree of amino acid identity and similarity of PpSFL-1and other homologous proteins (Pairwise comparison program was used: gappenalty: 10; gap extension penalty: 0.1; score matrix: blosum62)Swiss-Prot # Q59965 Q9L4T2 O22455 O22056 Q9MTH3 Protein Rna Rna Rna RnaRna name polymerase polymerase polymerase polymerase polymerase sigmafactor sigma factor sigma factor sigma factor sigma factor SpeciesSynechococcus Nostoc Arabidopsis Arabidopsis Sinapis alba sp.punctiforme thaliana thaliana (White (Mouse-ear (Mouse-ear mustard)cress) cress) Identity % 49% 49% 32% 42% 30% Similarity % 62% 61% 44%59% 42%

[0160] TABLE 9 Degree of amino acid identity and similarity of PpMYB-1and other homologous proteins (Pairwise comparison program was used: gappenalty: 10; gap extension penalty: 0.1; score matrix: blosum62).Swiss-Prot # Q9LLM9 Q9ZTD9 Q9SEZ4 Q9ZTD7 Q9MBG3 Protein name Myb-likePutative Putative myb Putative Myb protein transcription familytranscription transcription factor transcription factor factor-likefactor protein Species Oryza sativa Arabidopsis Arabidopsis ArabidopsisArabidopsis (Rice) thaliana thaliana thaliana thaliana (Mouse-ear(Mouse-ear (Mouse-ear cress) (Mouse-ear cress) cress) cress) Identity %37% 37% 32% 36% 29% Similarity % 47% 44% 38% 44% 37%

Example 6

[0161] Cloning of the Full-Length Physcomitrella patens cDNA Encodingfor APS-2, ZF-2, ZF-3, ZF-4, ZF-5, MYB-1, CABF-3 and SFL-1

[0162] Full-length clones corresponding to CABF-3 (SEQ ID NO:15) andAPS-2 (SEQ ID NO:9) were obtained by performing polymerase chainreaction (PCR) with gene-specific primers (see Table 10) and theoriginal. EST as the template since they were full-length. Theconditions for the reaction are described below under “Full-lengthAmplification.”

[0163] To isolate the clones encoding for PpZF-2, PpZF-3, PpZF-4, PpZF-5PpAPS-1, PpSFL-1 and PpMYB-1 from Physcomitrella patens, cDNA librarieswere created with SMART RACE cDNA Amplification kit (ClontechLaboratories) following the manufacturer's instructions. Total RNAisolated as described in Example 3 was used as the template. Thecultures were treated prior to RNA isolation as follows: Salt Stress: 2,6, 12, 24, 48 hours with 1-M NaCl-supplemented medium; Cold Stress: 4°C. for the same time points as for salt; Drought Stress: cultures wereincubated on dry filter paper for the same time points above. RNA wasthen pulled and used for isolation.

[0164] 5′ RACE Protocol

[0165] The EST sequences PpZF-2 (SEQ ID NO:2), PpZF-3 (SEQ ID NO:3),PpZF-4 (SEQ ID NO:4), PpZF-5 (SEQ ID NO:5), PpMYB-1 (SEQ ID NO:6) andPpSFL-1 (SEQ ID NO:8) identified from the database search as describedin Example 5 were used to design oligos for RACE (see Table 1). Theextended sequences for these genes were obtained by performing RapidAmplification of cDNA Ends polymerase chain reaction (RACE PCR) usingthe Advantage 2 PCR kit (Clontech Laboratories) and the SMART RACE cDNAamplification kit (Clontech Laboratories) using a Biometra T3Thermocycler following the manufacturer's instructions.

[0166] The sequences obtained from the RACE reactions contained the 5′end of the full-length coding regions of for PpZF-2, PpZF-3, PpZF-4,PpZF-5 PpAPS-1, PpSFL-1 and PpMYB-1 and were used to design oligos forfull-length cloning of the respective genes (see below under“Full-length Amplification).

[0167] Full-length Amplification

[0168] Full-length clones corresponding to PpCABF-3 (SEQ ID NO:15) andPpAPS-2 (SEQ ID NO:9) were obtained by performing polymerase chainreaction (PCR) with gene-specific primers (see Table 10) and theoriginal EST as the template. The conditions for the reaction werestandard conditions with PWO DNA polymerase (Roche). PCR was performedaccording to standard conditions and to manufacture's protocols(Sambrook et al. 1989. Molecular Cloning, A Laboratory Manual. 2ndEdition. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y.,Biometra T3 Thermocycler). The parameters for the reaction were: fiveminutes at 94° C. followed by five cycles of one minute at 94° C., oneminute at 50° C. and 1.5 minutes at 72° C. This was followed by twentyfive cycles of one minute at 94° C., one minute at 65° C. and 1.5minutes at 72° C.

[0169] Full-length clones for PpZF-2 (SEQ ID NO:10), PpZF-3 (SEQ IDNO:11), PpZF-4 (SEQ ID NO:12), PpZF-5 (SEQ ID NO:13), PpMYB-1 (SEQ IDNO:14) and PpSFL-1 (SEQ ID NO:16) and were isolated by repeating theRACE method but using the gene-specific primers as given in Table 10.

[0170] The amplified fragments were extracted from agarose gel with aQIAquick Gel Extraction Kit (Qiagen) and ligated into the TOPO pCR 2.1vector (Invitrogen) following manufacture's instructions. Recombinantvectors were transformed into Top10 cells (Invitrogen) using standardconditions (Sambrook et al. 1989. Molecular Cloning, A LaboratoryManual. 2nd Edition. Cold Spring Harbor Laboratory Press. Cold SpringHarbor, N.Y.). Transformed cells were selected for on LB agar containing100 μg/ml carbenicillin, 0.8 mg X-gal(5-bromo-4-chloro-3-indolyl-β-D-galactoside) and 0.8 mg IPTG(isopropylthio-β-D-galactoside) grown overnight at 37° C. White colonieswere selected and used to inoculate 3 ml of liquid LB containing 100μg/ml ampicillin and grown overnight at 37° C. Plasmid DNA was extractedusing the QIAprep Spin Miniprep Kit (Qiagen) following manufacture'sinstructions. Analyses of subsequent clones and restriction mapping wasperformed according to standard molecular biology techniques (Sambrooket al. 1989. Molecular Cloning, A Laboratory Manual. 2nd Edition. ColdSpring Harbor Laboratory Press. Cold Spring Harbor, N.Y.). Table 10Sites in the final Isolation Gene product Method Primers Race PrimerFull-length PCR PpCABF-3 XmaI/SacI PCR of N/A RC405 (SEQ ID NO:28)original EST ATCCCGGGCAGCGAG clone CACACAGCTAGCAAC TCTT RC406 (SEQ IDNO:29) GCGAGCTCACTCCCT CACGCGGTTGACAAT CT PpZF-2 XmaI/SacI 5′ RACE RC189RC606 (SEQ ID NO:31) and RT- (SEQ ID NO:30) ATCCCGGGAGGAAGC PCR forTGGCGGCCTC TGTCAGGGAAGAGAT Full-length GGTCTTCTTC GGA clone TCAGT RC607(SEQ ID NO:32) GCGAGCTCTGGCCGT AAAATCAGTTGTGGC GCTT PpZF-3 XmaI/ 5′ RACERC188 RC604 (SEQ ID NO:34) EcoRV andRT- (SEQ ID NO:33) ATCCCGGGAGGAGGPCR for CAGCGAAGCC ACTTGCGGAATGCAA Full-length CAATCGGGAT ATC cloneCAGGA RC605 (SEQ ID NO:35) GCGATATCCACCTGC TTCCACTCTCTACTTA TG PpZF-4XmaI/SacI 5′ RACE RC185 RC564 (SEQ ID NO:37) and RT- (SEQ ID NO:36)ATCCCGGGCACCAGT PCR for GACACCCGAT CCCGCTTAGTGTGTG Full-lengthTGAGCCGGCA TGT clone AGACG RC565 (SEQ ID NO:38) GCGAGGTCTTGATGCGACTCGCTCTCTCGA T PpZF-5 XmaI/SacI 5′ RACE RC187 RC612 (SEQ ID NO:40)and RT- (SEQ ID NO:39) ATCCCGGGTATCGAT PCR for CGGCGAGTGCCTGGAGCCCGTTGCA Full-length AGCAGCTTCT A clone AGAACG RC613 (SEQ IDNO:41) GCGAGCTCCTCCAAA GGACTTTGAAATATA GC PpAPS-2 EcoRV/ PCR of N/ARC395 (SEQ ID NO:42) SacI original EST GATATCGGAAGAAG cloneAATCCAAGGGAATGC GGTT RC396 (SEQ ID NO:43) GCGAGCTCTATGCTTCCGTGGGAGGAGCTT CAC PpSFL-1 XmaI/SacI 5′ RACE RC172 RC884 (SEQ ID NO:46)and RT- (SEQ ID NO:44) ATCCCGGGCTCGGAA PCR for CCGGCTGGGTGGACTGTGCATTGTC Full-length TGCCTCAGCT GA clone TGCGCA RC885 (SEQ IDNO:47) RC538 GCGAGCTCGCAGCAG (SEQ ID NO:45) AAGAAATCCACTTCT CGCTCCATCGGGT AACCTGGTGC CTTTGC PpMYB-1 SmaI/SmaI 5′ RACE RC170 RC701 (SEQ IDNO:49) and RT- (SEQ ID NO:48) ATCCCGGGCTGTTGT PCR for GGGTGCCGGTGTACAGTCTGTGGA Full-length TGATGCGAGG clone GTCCAG RC702 (SEQ ID NO:50)ATCCCGGGCTCACGG AGTAAAGGCCGTACC TT

Example 7

[0171] Engineering Stress-Tolerant Arabidopsis Plants by Over-Expressingthe Genes APS-2, ZF-2, ZF-3, ZF-4, ZF-5, MYB-1, CABF-3 and SFL-1

[0172] Binary Vector Construction:

[0173] The plasmid construct pACGH101 was digested with PstI (Roche) andFseI (NEB) according to manufacturers' instructions. The fragment waspurified by agarose gel and extracted via the Qiaex II DNA Extractionkit (Qiagen). This resulted in a vector fragment with the ArabidopsisActin2 promoter with internal intron and the OCS3 terminator. Primersfor PCR amplification of the NPTII gene were designed as follows:5′NPT-Pst: GCG-CTG-CAG-ATT-TCA-TTT-GGA-GAG-GAC-ACG (SEQ ID NO:51)3′NPT-Fse: CGC-GGC-CGG-CCT-CAG-AAG-AAC-TCG-TCA-AGA-AGG-CG. (SEQ IDNO:52)

[0174] The 0.9 kilobase NPTII gene was amplified via PCR from pCambia2301 plasmid DNA (94° C. for 60 seconds, {94° C. for 60 seconds, 61° C.(−0.1° C. per cycle) for 60 seconds, 72° C. for 2 minutes)×25 cycles,72° C. for 10 minutes on Biometra T-Gradient machine), and purified viathe Qiaquick PCR Extraction kit (Qiagen) as per manufacturer'sinstructions. The PCR DNA was then subcloned into the pCR-BluntII TOPOvector (Invitrogen) pursuant to the manufacturer's instructions(NPT-Topo construct). These ligations were transformed into Top10 cells(Invitrogen) and grown on LB plates with 50 μg/ml kanamycin sulfateovernight at 37° C. Colonies were then used to inoculate 2 ml LB mediawith 50 μg/ml kanamycin sulfate and grown overnight at 37° C. PlasmidDNA was recovered using the Qiaprep Spin Miniprep kit (Qiagen) andsequenced in both the 5′ and 3′ directions using standard conditions.Subsequent analysis of the sequence data using Vector NTI softwarerevealed no PCR errors present in the NPTII gene sequence.

[0175] The NPT-Topo construct was then digested with PstI (Roche) andFseI (NEB) according to manufacturers' instructions. The 0.9 kilobasefragment was purified on agarose gel and extracted by Qiaex II DNAExtraction kit (Qiagen). The Pst/Fse insert fragment from NPT-Topo andthe Pst/Fse vector fragment from pACGH101 were then ligated togetherusing T4 DNA Ligase (Roche) following manufacturer's instructions. Theligation was then transformed into Top10 cells (Invitrogen) understandard conditions, creating pBPSSC019 construct. Colonies wereselected on LB plates with 50 μg/ml kanamycin sulfate and grownovernight at 37° C. These colonies were then used to inoculate 2 ml LBmedia with 50 μg/ml kanamycin sulfate and grown overnight at 37° C.Plasmid DNA was recovered using the Qiaprep Spin Miniprep kit (Qiagen)following the manufacturer's instructions.

[0176] The pBPSSC019 construct was digested with KpnI and BsaI (Roche)according to manufacturer's instructions. The fragment was purified viaagarose gel and then extracted via the Qiaex II DNA Extraction kit(Qiagen) as per its instructions, resulting in a 3 kilobase Act-NPTcassette, which included the Arabidopsis Actin2 promoter with internalintron, the NPTII gene and the OCS3 terminator.

[0177] The pBPSJH001 vector was digested with SpeI and ApaI (Roche) andblunt-end filled with Klenow enzyme and 0.1 mM dNTPs (Roche) accordingto manufacture's instructions. This produced a 10.1 kilobase vectorfragment minus the Gentamycin cassette, which was recircularized byself-ligating with T4 DNA Ligase (Roche), and transformed into Top10cells (Invitrogen) via standard conditions. Transformed cells wereselected for on LB agar containing 50 μg/ml kanamycin sulfate and grownovernight at 37° C. Colonies were then used to inoculate 2 ml of liquidLB containing 50 μg/ml kanamycin sulfate and grown overnight at 37° C.Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen)following manufacture's instructions. The recircularized plasmid wasthen digested with KpnI (Roche) and extracted from agarose gel via theQiaex II DNA Extraction kit (Qiagen) as per manufacturer's instructions.

[0178] The Act-NPT Kpn-cut insert and the Kpn-cut pBPSJH001recircularized vector were then ligated together using T4 DNA Ligase(Roche) and transformed into Top10 cells (Invitrogen) as permanufacturers' instructions. The resulting construct, pBPSSC022, nowcontained the Super Promoter, the GUS gene, the NOS terminator, and theAct-NPT cassette. Transformed cells were selected for on LB agarcontaining 50 μg/ml kanamycin sulfate and grown overnight at 37° C.Colonies were then used to inoculate 2 ml of liquid LB containing 50μg/ml kanamycin sulfate and grown overnight at 37° C. Plasmid DNA wasextracted using the QIAprep Spin Miniprep Kit (Qiagen) followingmanufacturer's instructions. After confirmation of ligation success viarestriction digests, pBPSSC022 plasmid DNA was further propagated andrecovered using the Plasmid Midiprep Kit (Qiagen) following themanufacturer's instructions.

[0179] Subcloning of APS-2, ZF-2, ZF-3, ZF-4, ZF-5, MYB-1, CABF-3 andSFL-1 into the Binary Vector

[0180] The fragments containing the different Physcomitrella patenstranscription factors were subcloned from the recombinant PCR2.1 TOPOvectors by double digestion with restriction enzymes (see Table 11)according to manufacturer's instructions. The subsequence fragment wasexcised from agarose gel with a QIAquick Gel Extraction Kit (QIAgen)according to manufacture's instructions and ligated into the binaryvectors pBPSSC022, cleaved with XmaI and Ecl136II and dephosphorylatedprior to ligation. The resulting recombinant pBPSSC022 contained thecorresponding transcription factor in the sense orientation under theconstitutive super promoter. TABLE 11 Listed are the names of thevarious constructs of the Physcomitrella patens transcription factorsused for plant transformation Enzymes used to generate gene Enzymes usedto Binary Vector Gene fragment restrict pBPSJH001 Construct PpCABF-3XmaI/SacI XmaI/SacI pBPSLVM185 PpZF-2 XmaI/SacI XmaI/SacI pBPSSY008PpZF-3 XmaI/ XmaI/Ec1136 pBPSSY017 EcoRV PpZF-4 XmaI/SacI XmaI/SacIpBPSLVM163 PpZF-5 XmaI/SacI XmaI/SacI pBPSERG006 PpAPS-2 EcoRV/SacISmaI/SacI pBPSLVM161 PpSFL-1 XmaI/SacI XmaI/SacI pBPSERG001 PpMYB-1SmaI/SmaI SmaI/Ec1136 pBPSERG020

[0181] Agrobacterium Transformation

[0182] The recombinant vectors were transformed into Agrobacteriumtumefaciens C58C1 and PMP90 according to standard conditions (Hoefgenand Willmitzer, 1990).

[0183] Plant Transformation

[0184]Arabidopsis thaliana ecotype C24 were grown and transformedaccording to standard conditions (Bechtold 1993, Acad. Sci. Paris.316:1194-1199; Bent et al. 1994, Science 265:1856-1860).

[0185] Screening of Transformed Plants

[0186] T1 seeds were sterilized according to standard protocols (Xionget al. 1999, Plant Molecular Biology Reporter 17: 159-170). Seeds wereplated on {fraction (1/2)} Murashige and Skoog media (MS)(Sigma-Aldrich) pH 5.7 with KOH, 0.6% agar and supplemented with 1%sucrose, 0.5 g/L 2-[N-Morpholino]ethansulfonic acid (MES)(Sigma-Aldrich), 50 μg/ml kanamycin (Sigma-Aldrich), 500 μg/mlcarbenicillan (Sigma-Aldrich) and 2 μg/ml benomyl (Sigma-Aldrich). Seedson plates were vernalized for four days at 4° C. The seeds weregerminated in a climatic chamber at an air temperature of 22° C. andlight intensity of 40 micromols^(−1m2) (white light; Philips TL 65W/25fluorescent tube) and 16 hours light and 8 hours dark day length cycle.Transformed seedlings were selected after 14 days and transferred to ½MS media pH 5.7 with KOH 0.6% agar plates supplemented with 0.6% agar,1% sucrose, 0.5 g/L MES (Sigma-Aldrich), and 2 μg/ml benomyl(Sigma-Aldrich) and allowed to recover for five-seven days.

[0187] Drought Tolerance Screening

[0188] T1 seedlings were transferred to dry, sterile filter paper in apetri dish and allowed to desiccate for two hours at 80% RH (relativehumidity) in a Percival Growth CU3615, micromols^(−1m2) (white light;Philips TL 65W/25 fluorescent tube). The RH was then decreased to 60%and the seedlings were desiccated further for eight hours. Seedlingswere then removed and placed on ½ MS 0.6% agar plates supplemented with2 μg/ml benomyl (Sigma-Aldrich) and 0.5 g/L MES ((Sigma-Aldrich) andscored after five days.

[0189] Under drought stress conditions, PpCABF-3 over-expressingArabidopsis thaliana plants showed an 70% (39 survivors from 56 stressedplants) survival rate to the stress screening; PpZF-2, 98% (39 survivorsfrom 40 stressed plants); PpZF-3, 94% (59 survivors from 63 stressedplants); PpZF-4, 94% (16 survivors from 17 stressed plants); PpZF-5, 80%(8 survivors from 10 stressed plants); PpAPS-265% (13 survivors from 20stressed plants); and PpMYB-180% (8 survivors from 10 stressed plants);whereas the untransformed control a 28% (16 survivors from 57 stressedplants) survival rate. It is noteworthy that the analyses of thesetransgenic lines were performed with T1 plants, and therefore, theresults will be better when a homozygous, strong expresser is found.TABLE 12 Summary of the drought stress tests Drought Stress Test Numberof Total number of Percentage of Gene Name survivors plants survivorsPpCABF-3 39 56 70% PpZF-2 39 40 98% PpZF-3 59 63 94% PpZF-4 16 17 94%PpZF-5 8 10 80% PpAPS-2 13 20 65 PpMYB-1 8 10 80% Control 16 57 28%

[0190] Freezing Tolerance Screening

[0191] Seedlings were moved to petri dishes containing ½ MS 0.6% agarsupplemented with 2% sucrose and 2 μg/ml benomyl. After four days, theseedlings were incubated at 4° C. for 1 hour and then covered withshaved ice. The seedlings were then placed in an EnvironmentalSpecialist ES2000 Environmental Chamber and incubated for 3.5 hoursbeginning at −1.0° C. decreasing −1° C. hour. The seedlings were thenincubated at −5.0° C. for 24 hours and then allowed to thaw at 5° C. for12 hours. The water was poured off and the seedlings were scored after 5days.

[0192] Under freezing stress conditions, PpCABF-3 over-expressingArabidopsis thaliana plants showed an 98% (41 survivors from 42 stressedplants) survival rate to the stress screening; PpZF-2, 86% (19 survivorsfrom 22 stressed plants); and PpZF-3, 74% (14 survivors from 19 stressedplants); whereas the untransformed control a 28% (16 survivors from 57stressed plants) survival rate. It is noteworthy that the analyses ofthese transgenic lines were performed with T1 plants, and therefore, theresults will be better when a homozygous, strong expresser is found.TABLE 13 Summary of the freezing stress tests Freezing Stress Test Totalnumber of Percentage of Gene Name Number of survivors plants survivorsPpCABF-3 41 42 98% PpZF-2 19 22 86% PpZF-3 14 19 74% Control 1 48 2%

[0193] Salt Tolerance Screening

[0194] Seedlings were transferred to filter paper soaked in ½ MS andplaced on 12 MS 0.6% agar supplemented with 2 μg/ml benomyl the nightbefore the salt tolerance screening. For the salt tolerance screening,the filter paper with the seedlings was moved to stacks of sterilefilter paper, soaked in 50 mM NaCl, in a petri dish. After two hours,the filter paper with the seedlings was moved to stacks of sterilefilter paper, soaked with 200 mM NaCl, in a petri dish. After two hours,the filter paper with the seedlings was moved to stacks of sterilefilter paper, soaked in 600 mM NaCl, in a petri dish. After 10 hours,the seedlings were moved to petri dishes containing ½ MS 0.6% agarsupplemented with 2 μg/ml benomyl. The seedlings were scored after 5days.

[0195] The transgenic plants are screened for their improved salttolerance demonstrating that transgene expression confers salttolerance.

Example 8

[0196] Detection of the APS-2, ZF-2, ZF-3, ZF-4, ZF-5, MYB-1, CABF-3 andSFL-1 Transgenes in the Transgenic Arabidopsis Lines

[0197] One leaf from a wild type and a transgenic Arabidopsis plant washomogenized in 250 μl Hexadecyltrimethyl ammonium bromide (CTAB) buffer(2% CTAB, 1.4 M NaCl, 8 mM EDTA and 20 mM Tris pH 8.0) and 1 μlβ-mercaptoethanol. The samples were incubated at 60-65° C. for 30minutes and 250 μl of Chloroform was then added to each sample. Thesamples were vortexed for 3 minutes and centrifuged for 5 minutes at18,000×g. The supernatant was taken from each sample and 150 μlisopropanol was added. The samples were incubated at room temperaturefor 15 minutes, and centrifuged for 10 minutes at 18,000×g. Each pelletwas washed with 70% ethanol, dried, and resuspended in 20 μl TE. 4 μl ofabove suspension was used in a 20 μl PCR reaction using Taq DNApolymerase (Roche Molecular Biochemicals) according to themanufacturer's instructions. Binary vector plasmid with each gene clonedin was used as positive control, and the wild type C24 genomic DNA wasused as negative control in the PCR reactions. 10 μl PCR reaction wasanalyzed on 0.8% agarose/ethidium bromide gel. The PCR program used wasas follows: 30 cycles of 1 minute at 94° C., 1 minute at 62° C. and 4minutes at 70° C., followed by 10 minutes at 72° C. The following primerwas used as 5′ primer: Bfwd: 5′GCTGACACGCCAAGCCTCGCTAGTC3′. (SEQ IDNO:53) The gene-specific primers and the size of the amplified bands(Gene Product Size) are listed below.

[0198] PpCABF-3

[0199] Primer: RC406: GCGAGCTCACTCCCTCACGCGGTTGACAATCT

[0200] Gene Product Size: 800 bp (SEQ ID NO:54)

[0201] PpZF-2

[0202] Primer: RC607: GCGAGCTCTGGCCGTAAAATCAGTTGTGGCGCTT

[0203] Gene Product Size: 1800 bp (SEQ ID NO:55)

[0204] PpZF-3

[0205] Primer: RC605: GCGATATCCACCTGCTTCCACTCTCTACTTATG

[0206] Gene Product Size: 2000 bp (SEQ ID NO:56)

[0207] PpZF-4

[0208] Primer: RC565: GCGAGCTCTTGATGCGACTCGCTCTCTCGAT

[0209] Gene Product Size: 800 bp (SEQ ID NO:57)

[0210] PpZF-5

[0211] Primer: RC613: GCGAGCTCCTCCAAAGGACTTTGAAATATAGC

[0212] Gene Product Size: 2700 bp (SEQ ID NO:58)

[0213] PpAPS-2

[0214] Primer: RC396: GCGAGCTCTATGCTTCCGTGGGAGGAGCTTCAC

[0215] Gene Product Size: 1000 bp (SEQ ID NO:59)

[0216] PpSFL-1

[0217] Primer: RC885: GCGAGCTCGCAGCAGAAGAAATCCACTTCTGGT

[0218] Gene Product Size: 1700 bp (SEQ ID NO:60)

[0219] PpMYB-1

[0220] Primer: RC702: ATCCCGGGCTCACGGAGTAAAGGCCGTACCTT

[0221] Gene Product Size: 2400 bp (SEQ ID NO:61)

[0222] The transgenes were successfully amplified from the T1 transgeniclines, but not from the wild type C24. This result indicates that the T1transgenic plants contain at least one copy of the transgenes. There wasno indication of existence of either identical or very similar genes inthe untransformed Arabidopsis thaliana control which could be amplifiedby this method.

Example 9

[0223] Detection of the APS-2, ZF-2, ZF-3, ZF-4, ZF-5, MYB-1, CABF-3 andSFL-1 Transgene mRNA in Transgenic Arabidopsis Lines

[0224] Transgene expression was detected using RT-PCR. Total RNA wasisolated from stress-treated plants using a procedure adapted from(Verwoerd et al., 1989 NAR 17:2362). Leaf samples (50-100 mg) werecollected and ground to a fine powder in liquid nitrogen. Ground tissuewas resuspended in 500 μl of a 80° C., 1:1 mixture, of phenol toextraction buffer (100 mM LiCl, 100 mM Tris pH8, 10 mM EDTA, 1% SDS),followed by brief vortexing to mix. After the addition of 250 μl ofchloroform, each sample was vortexed briefly. Samples were thencentrifuged for 5 minutes at 12,000×g. The upper aqueous phase wasremoved to a fresh eppendorf tube. RNA was precipitated by adding{fraction (1/10)}^(th) volume 3M sodium acetate and 2 volumes 95%ethanol. Samples were mixed by inversion and placed on ice for 30minutes. RNA was pelleted by centrifugation at 12,000×g for 10 minutes.The supernatant was removed and pellets briefly air-dried. RNA samplepellets were resuspended in 10 μl DEPC treated water.

[0225] To remove contaminating DNA from the samples, each was treatedwith RNase-free DNase (Roche) according to the manufacturer'srecommendations. cDNA was synthesized from total RNA using the 1^(st)Strand cDNA synthesis kit (Boehringer Mannheim) following manufacturer'srecommendations. PCR amplification of a gene-specific fragment from thesynthesized cDNA was performed using Taq DNA polymerase (Roche) andgene-specific primers (see Table 4 for primers) in the followingreaction: 1×PCR buffer, 1.5 mM MgCl₂, 0.2 μM each primer, 0.2 μM dNTPs,1 unit polymerase, 5 μl cDNA from synthesis reaction. Amplification wasperformed under the following conditions: Denaturation, 95° C., 1minute; annealing, 62° C., 30 seconds; extension, 72° C., 1 minute, 35cycles; extension, 72° C., 5 minutes; hold, 4° C., forever. PCR productswere run on a 1% agarose gel, stained with ethidium bromide, andvisualized under UV light using the Quantity-One gel documentationsystem (Bio-Rad). Expression of the transgenes was detected in the T1transgenic line.

[0226] These results indicated that the transgenes are expressed in thetransgenic lines and strongly suggested that their gene product improvedplant stress tolerance in the transgenic lines. In agreement with theprevious statement, no expression of identical or very similarendogenous genes could be detected by this method. These results are inagreement with the data from Example 7. TABLE 14 Gene 5′ primer3′ primer PpCABF-2 RC405: (SEQ ID NO:62) RC406: (SEQ ID ATCCCGGGCAGCGAGCNO:63) ACACAGCTAGCAACTC GCGAGCTCACTCCCTC TT ACGCGGTTGACAATCT PpZF-2RC1191: (SEQ ID NO:64) RC1192: (SEQ ID GCCCGTTGTGTCGCAC NO:65)GAGTGTGGGA GCCGCTGGACCAGACC TCGGAATGT PpZF-3 RC1203: (SEQ ID NO:66)RC1204: (SEQ ID GAGGCAGTCATGCAAT NO:67) CGACCCCAA GCGAAGCCCAATCGGGATCAGCAGCA PpZF-4 RC564: (SEQ ID NO:68) RC565: (SEQ ID ATCCCGGGCACCAGTCNO:69) CCGCTTAGTGTGTGTGT GCGAGCTCTTGATGCG ACTCGCTCTCTCGAT PpZF-5 RG1209:(SEQ ID NO:70) RC1210: (SEQ ID CGCATCGCATCTGGCG NO:71) AACTTTGTG3′primer for EST281 at#1368 GC = 58% CGTACCACGATTGCTCT AGCGCACGT PpAPS-1RC395: (SEQ ID NO:72) RC396: (SEQ ID GCGATATCGGAAGAAG NO:73)AATCCAAGGGAATGCG GCGAGCTCTATGCTTCC GTT GTGGGAGGAGCTTCAC PpAPS- RC405:(SEQ ID NO:74) RC406: (SEQ ID ATCCCGGGCAGCGAGC NO:75) ACACAGCTAGCAACTCGCGAGCTCACTCCCTC TT ACGCGGTTGACAATCT PpSFL-1 RC1191: (SEQ ID NO:76)RC1192: (SEQ ID GCCCGTTGTGTCGCAC NO:77) GAGTGTGGGA GCCGCTGGACCAGACCTCGGAATGT PpMYB-1 RC1203: (SEQ ID NO:78) RC1204: (SEQ IDGAGGCAGTCATGCAAT NO:79) CGACCCCAA GCGAAGCCCAATCGGG ATCAGCAGCA

Example 10

[0227] Engineering Stress-Tolerant Soybean Plants by Over-Expressing theAPS-2, ZF-2, ZF-3, ZF-4, ZF-5, MYB-1, CABF-3 and SFL-1 Gene

[0228] The constructs pBPSLVM185, pBPSSY008, pBPSSY017, pBPSLVM163,pBPSERG006, pBPSLVM161, pBPSERG001 and pBPSERG020 were used to transformsoybean as described below.

[0229] Seeds of soybean were surface sterilized with 70% ethanol for 4minutes at room temperature with continuous shaking, followed by 20%(v/v) Clorox supplemented with 0.05% (v/v) Tween for 20 minutes withcontinuous shaking. Then, the seeds were rinsed 4 times with distilledwater and placed on moistened sterile filter paper in a Petri dish atroom temperature for 6 to 39 hours. The seed coats were peeled off, andcotyledons are detached from the embryo axis. The embryo axis wasexamined to make sure that the meristematic region is not damaged. Theexcised embryo axes were collected in a half-open sterile Petri dish andair-dried to a moisture content less than 20% (fresh weight) in a sealedPetri dish until further use.

[0230]Agrobacterium tumefaciens culture was prepared from a singlecolony in LB solid medium plus appropriate antibiotics (e.g. 100 mg/lstreptomycin, 50 mg/l kanamycin) followed by growth of the single colonyin liquid LB medium to an optical density at 600 nm of 0.8. Then, thebacteria culture was pelleted at 7000 rpm for 7 minutes at roomtemperature, and resuspended in MS (Murashige and Skoog, 1962) mediumsupplemented with 100 μM acetosyringone. Bacteria cultures wereincubated in this pre-induction medium for 2 hours at room temperaturebefore use. The axis of soybean zygotic seed embryos at approximately15% moisture content were imbibed for 2 hours at room temperature withthe pre-induced Agrobacterium suspension culture. The embryos areremoved from the imbibition culture and were transferred to Petri dishescontaining solid MS medium supplemented with 2% sucrose and incubatedfor 2 days, in the dark at room temperature. Alternatively, the embryoswere placed on top of moistened (liquid MS medium) sterile filter paperin a Petri dish and incubated under the same conditions described above.After this period, the embryos were transferred to either solid orliquid MS medium supplemented with 500 mg/L carbenicillin or 300 mg/Lcefotaxime to kill the agrobacteria. The liquid medium was used tomoisten the sterile filter paper. The embryos were incubated during 4weeks at 25° C., under 150 μmol m⁻²sec⁻¹ and 12 hours photoperiod. Oncethe seedlings produced roots, they were transferred to sterile metromixsoil. The medium of the in vitro plants was washed off beforetransferring the plants to soil. The plants were kept under a plasticcover for 1 week to favor the acclimatization process. Then the plantswere transferred to a growth room where they were incubated at 25° C.,under 150 μmol m⁻² sec⁻¹ light intensity and 12 hours photoperiod forabout 80 days.

[0231] The transgenic plants were then screened for their improveddrought, salt and/or cold tolerance according to the screening methoddescribed in Example 7 demonstrating that transgene expression confersstress tolerance.

Example 11

[0232] Engineering Stress-Tolerant Rapeseed/Canola Plants byOver-Expressing the APS-2, ZF-2, ZF-3, ZF-4, ZF-5, MYB-1, CABF-3 andSFL-1 Genes

[0233] The constructs pBPSLVM185, pBPSSY008, pBPSSY017, pBPSLVM163,pBPSERG006, pBPSLVM161, pBPSERG001 and pBPSERG020 were used to transformrapseed/canola as described below.

[0234] The method of plant transformation described herein is alsoapplicable to Brassica and other crops. Seeds of canola are surfacesterilized with 70% ethanol for 4 minutes at room temperature withcontinuous shaking, followed by 20% (v/v) Clorox supplemented with 0.05%(v/v) Tween for 20 minutes, at room temperature with continuous shaking.Then, the seeds are rinsed 4 times with distilled water and placed onmoistened sterile filter paper in a Petri dish at room temperature for18 hours. Then the seed coats are removed and the seeds are air driedovernight in a half-open sterile Petri dish. During this period, theseeds lose approx. 85% of its water content. The seeds are then storedat room temperature in a sealed Petri dish until further use. DNAconstructs and embryo imbibition are as described in Example 10. Samplesof the primary transgenic plants (T0) are analyzed by PCR to confirm thepresence of T-DNA. These results are confirmed by Southern hybridizationin which DNA is electrophoresed on a 1% agarose gel and transferred to apositively charged nylon membrane (Roche Diagnostics). The PCR DIG ProbeSynthesis Kit (Roche Diagnostics) is used to prepare adigoxigenin-labelled probe by PCR, and used as recommended by themanufacturer.

[0235] The transgenic plants are then screened for their improved stresstolerance according to the screening method described in Example 7demonstrating that transgene expression confers drought tolerance.

Example 12

[0236] Engineering Stress-Tolerant Corn Plants by Over-Expressing theAPS-2, ZF-2, ZF-3, ZF-4, ZF-5, MYB-1, CABF-3 or SFL-1 Genes

[0237] The constructs pBPSLVM185, pBPSSY008, pBPSSY017, pBPSLVM163,pBPSERG006, pBPSLVM161, pBPSERG001 and pBPSERG020 were used to transformcorn as described below.

[0238] Transformation of maize (Zea Mays L.) is performed with themethod described by Ishida et al. 1996. Nature Biotch 14745-50. Immatureembryos are co-cultivated with Agrobacterium tumefaciens that carry“super binary” vectors, and transgenic plants are recovered throughorganogenesis. This procedure provides a transformation efficiency ofbetween 2.5% and 20%. The transgenic plants are then screened for theirimproved drought, salt and/or cold tolerance according to the screeningmethod described in Example 7 demonstrating that transgene expressionconfers stress tolerance.

Example 13

[0239] Engineering Stress-Tolerant Wheat Plants by Over-Expressing theAPS-2, ZF-2, ZF-3, ZF-4, ZF-5, MYB-1, CABF-3 or SFL-1 Genes

[0240] The constructs pBPSLVM185, pBPSSY008, pBPSSY017, pBPSLVM163,pBPSERG006, pBPSLVM161, pBPSERG001, pBPSERG020 were used to transformwheat as described below.

[0241] Transformation of wheat is performed with the method described byIshida et al. 1996 Nature Biotch. 14745-50. Immature embryos areco-cultivated with Agrobacterium tumefaciens that carry “super binary”vectors, and transgenic plants are recovered through organogenesis. Thisprocedure provides a transformation efficiency between 2.5% and 20%. Thetransgenic plants are then screened for their improved stress toleranceaccording to the screening method described in Example 7 demonstratingthat transgene expression confers drought tolerance.

Example 14

[0242] Identification of Homologous and Heterologous Genes

[0243] Gene sequences can be used to identify homologous or heterologousgenes from cDNA or genomic libraries. Homologous genes (e.g. full-lengthcDNA clones) can be isolated via nucleic acid hybridization using forexample cDNA libraries. Depending on the abundance of the gene ofinterest, 100,000 up to 1,000,000 recombinant bacteriophages are platedand transferred to nylon membranes. After denaturation with alkali, DNAis immobilized on the membrane by e.g. UV cross linking. Hybridizationis carried out at high stringency conditions. In aqueous solutionhybridization and washing is performed at an ionic strength of 1 M NaCland a temperature of 68° C. Hybridization probes are generated by e.g.radioactive (³²P) nick transcription labeling (High Prime, Roche,Mannheim, Germany). Signals are detected by autoradiography.

[0244] Partially homologous or heterologous genes that are related butnot identical can be identified in a manner analogous to theabove-described procedure using low stringency hybridization and washingconditions. For aqueous hybridization, the ionic strength is normallykept at 1 M NaCl while the temperature is progressively lowered from 68to 42° C.

[0245] Isolation of gene sequences with homologies (or sequenceidentity/similarity) only in a distinct domain of (for example 10-20amino acids) can be carried out by using synthetic radio labeledoligonucleotide probes. Radio labeled oligonucleotides are prepared byphosphorylation of the 5-prime end of two complementary oligonucleotideswith T4 polynucleotide kinase. The complementary oligonucleotides areannealed and ligated to form concatemers. The double strandedconcatemers are than radiolabeled by, for example, nick transcription.Hybridization is normally performed at low stringency conditions usinghigh oligonucleotide concentrations.

[0246] Oligonucleotide Hybridization Solution:

[0247] 6×SSC

[0248] 0.01 M sodium phosphate

[0249] 1 mM EDTA (pH 8)

[0250] 0.5% SDS

[0251] 100 μg/ml denatured salmon sperm DNA

[0252] 0.1% nonfat dried milk

[0253] During hybridization, temperature is lowered stepwise to 5-10° C.below the estimated oligonucleotide Tm or down to room temperaturefollowed by washing steps and autoradiography. Washing is performed withlow stringency such as 3 washing steps using 4×SSC. Further details aredescribed by Sambrook, J. et al. (1989), “Molecular Cloning: ALaboratory Manual”, Cold Spring Harbor Laboratory Press or Ausubel, F.M. et al. (1994) “Current Protocols in Molecular Biology”, John Wiley &Sons.

Example 15

[0254] Identification of Homologous Genes by Screening ExpressionLibraries with Antibodies

[0255] c-DNA clones can be used to produce recombinant protein forexample in E. coli (e.g. Qiagen QIAexpress pQE system). Recombinantproteins are then normally affinity purified via Ni-NTA affinitychromatography (Qiagen). Recombinant proteins are then used to producespecific antibodies for example by using standard techniques for rabbitimmunization. Antibodies are affinity purified using a Ni-NTA columnsaturated with the recombinant antigen as described by Gu et al., 1994BioTechniques 17:257-262. The antibody can than be used to screenexpression cDNA libraries to identify homologous or heterologous genesvia an immunological screening (Sambrook, J. et al. (1989), “MolecularCloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press orAusubel, F. M. et al. (1994) “Current Protocols in Molecular Biology”,John Wiley & Sons).

Example 16

[0256] In Vivo Mutagenesis

[0257] In vivo mutagenesis of microorganisms can be performed by passageof plasmid (or other vector) DNA through E. coli or other microorganisms(e.g. Bacillus spp. or yeasts such as Saccharomyces cerevisiae) whichare impaired in their capabilities to maintain the integrity of theirgenetic information. Typical mutator strains have mutations in the genesfor the DNA repair system (e.g., mutHLS, mutD, mutT, etc.; forreference, see Rupp, W. D. (1996) DNA repair mechanisms, in: Escherichiacoli and Salmonella, p. 2277-2294, ASM: Washington.) Such strains arewell known to those skilled in the art. The use of such strains isillustrated, for example, in Greener, A. and Callahan, M. (1994)Strategies 7: 32-34. Transfer of mutated DNA molecules into plants ispreferably done after selection and testing in microorganisms.Transgenic plants are generated according to various examples within theexemplification of this document.

Example 17

[0258] In vitro Analysis of the Function of Physcomitrella Genes inTransgenic Organisms

[0259] The determination of activities and kinetic parameters of enzymesis well established in the art. Experiments to determine the activity ofany given altered enzyme must be tailored to the specific activity ofthe wild-type enzyme, which is well within the ability of one skilled inthe art. Overviews about enzymes in general, as well as specific detailsconcerning structure, kinetics, principles, methods, applications andexamples for the determination of many enzyme activities may be found,for example, in the following references: Dixon, M., and Webb, E. C.,(1979) Enzymes. Longmans: London; Fersht, (1985) Enzyme Structure andMechanism. Freeman: New York; Walsh, (1979) Enzymatic ReactionMechanisms. Freeman: San Francisco; Price, N. C., Stevens, L. (1982)Fundamentals of Enzymology. Oxford Univ. Press: Oxford; Boyer, P. D.,ed. (1983) The Enzymes, 3^(rd) ed. Academic Press: New York; Bisswanger,H., (1994) Enzymkinetik, 2^(nd) ed. VCH: Weinheim (ISBN 3527300325);Bergmeyer, H. U., Bergmeyer, J., Graβ1, M., eds. (1983-1986) Methods ofEnzymatic Analysis, 3^(rd) ed., vol. I-XII, Verlag Chemie: Weinheim; andUllmann's Encyclopedia of Industrial Chemistry (1987) vol. A9, Enzymes.VCH: Weinheim, p. 352-363.

[0260] The activity of proteins which bind to DNA can be measured byseveral well-established methods, such as DNA band-shift assays (alsocalled gel retardation assays). The effect of such proteins on theexpression of other molecules can be measured using reporter gene assays(such as that described in Kolmar, H. et al. (1995) EMBO J. 14:3895-3904 and references cited therein). Reporter gene test systems arewell known and established for applications in both pro- and eukaryoticcells, using enzymes such as β-galactosidase, green fluorescent protein,and several others.

[0261] The determination of activity of membrane-transport proteins canbe performed according to techniques such as those described in Gennis,R. B. Pores, Channels and Transporters, in Biomembranes, MolecularStructure and Function, pp. 85-137, 199-234 and 270-322, Springer:Heidelberg (1989).

Example 18

[0262] Purification of the Desired Product from Transformed Organisms

[0263] Recovery of the desired product from plant material (i.e.,Physcomitrella patents or Arabidopsis thaliana), fungi, algae, ciliates,C. glutamicum cells, or other bacterial cells transformed with thenucleic acid sequences described herein, or the supernatant of theabove-described cultures can be performed by various methods well knownin the art. If the desired product is not secreted from the cells, canbe harvested from the culture by low-speed centrifugation, the cells canbe lysed by standard techniques, such as mechanical force orsonification. Organs of plants can be separated mechanically from othertissue or organs. Following homogenization cellular debris is removed bycentrifugation, and the supernatant fraction containing the solubleproteins is retained for further purification of the desired compound.If the product is secreted from desired cells, then the cells areremoved from the culture by low-speed centrifugation, and the supernatefraction is retained for further purification.

[0264] The supernatant fraction from either purification method issubjected to chromatography with a suitable resin, in which the desiredmolecule is either retained on a chromatography resin while many of theimpurities in the sample are not, or where the impurities are retainedby the resin while the sample is not. Such chromatography steps may berepeated as necessary, using the same or different chromatographyresins. One skilled in the art would be well-versed in the selection ofappropriate chromatography resins and in their most efficaciousapplication for a particular molecule to be purified. The purifiedproduct may be concentrated by filtration or ultrafiltration, and storedat a temperature at which the stability of the product is maximized.

[0265] There is a wide array of purification methods known to the artand the preceding method of purification is not meant to be limiting.Such purification techniques are described, for example, in Bailey, J.E. & Ollis, D. F. Biochemical Engineering Fundamentals, McGraw-Hill: NewYork (1986). Additionally, the identity and purity of the isolatedcompounds may be assessed by techniques standard in the art. Theseinclude high-performance liquid chromatography (HPLC), spectroscopicmethods, staining methods, thin layer chromatography, NIRS, enzymaticassay, or microbiologically. Such analysis methods are reviewed in:Patek et al., 1994 Appl. Environ. Microbiol. 60:133-140; Malakhova etal., 1996 Biotekhnologiya 11:27-32; and Schmidt et al., 1998 BioprocessEngineer. 19:67-70. Ulmann's Encyclopedia of Industrial Chemistry,(1996) vol. A27, VCH: Weinheim, p. 89-90, p. 521-540, p. 540-547, p.559-566, 575-581 and p. 581-587; Michal, G. (1999) Biochemical Pathways:An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons;Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in:Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17.!APPENDIX Nucleotide sequence of the partial APS-2 from Physcomitrellapatens (SEQ ID NO:1)TCAAGCCACTCATCCGAGCATAGAACATCACAACCCACCTTGATGATCATTCTCTCAGCCGACCAGCGTCAATTACGCTGCGTATCGCTCTAGCTTGAGGAAGGCACCCTCGCCCTCTTCGCCGCGGAAGTAGCCCTCTGCTTCACGAGGGCGGCAAAACTCTCCCAAGGCAGTTCCGGGGGGATGGGATATAGCTGCAGCTGCTGTGGGGAATCCTCAAAATTGTAGGGGATCTTCTTCTTTGTGTAGAAGATGCCAACATCGTAGGCCCGGGCAGCTTCTTCCGGAGTTTCATATGTTCCCAGCCATATCTTACGTTTCTGAGATGTGGGTCGAATTTCTGTCACCCATTTGTTTAGCTCGGGCCGGTGCCGAACCCCCCTAAAACTGGTCGTATCGCCAGTGTTGCTAGCAGAAACTTCTTCGGTATCCCATGCCGATGGGGCCTTATTTAAATCAATATTCCGAAATTTAAAGGCATTCCGACCGCTAGTGTCTTTCGCCGCTAACCGCATTCCCTTGGATTCTTCTTCCAAACTAGATTCAGACTTGCTCTCCTGCCAACTTCTTTTTTCACTTTCGGGGATTCTATTTTAGTGGTTAACTGCAACGCCTGTTCTTTGACCTTGCCACCACAAGGATCCCACTTCTTTGTTTTGGGCTTCCCCTGTTCAATAATGCTGGAAATTGTCAAATTCATGAACTACCCAATTGCAACCCCTCCCACCGGGATGGATTGATCGCCAAAATTTCGTAGTAACTTAACTTTCATACAACAACTTGAGTTCCTTCGCTATTAGGGACACGTGGCAGAAACTTGGACGTGCAAGCGTATGTACTCATCAGAGTTTGACAGCGCATAAAATCATATAAAAAGTCTTGAAGAAGCGTTGTTTAATTCATGGGTAACCACGAGTTACGCGGAGCGTCGGCAGCAAGGAGAGGACGACCAGGCGGCAAGAAGATGCGTCGGCAAGAGCTCGTGC Nucleotide sequenceof the partial ZF-2 from Physcomitrella patens (SEQ ID NO:2)TTTTTTTTGGCGAAAATGGGTAAAAATTTCCGTGGCCGTAAAATCAGTTGTGGCGCTTGCCTTGCAATAAGCTGGTTATCGTAAAATGGCAATTACCTTGATATGTTCACTAGGTTCGTGTCAGTGAGATGCCTTGCAGAAGGCGATTTCCCGTTTATTTACAACTCTACATGTTACTGAAGCACTGTGGCATTCAATCTCCTAACCTAGAGATGCTTAACTGCTTCGTGCATGATCTATATTACATCTTGAGCCTCAGACTGTGGCGTCTTATTGCACATCTAGCGCTATATATTACACTACGGTACACCATCGGAAGAAGTGAAGAAGGAATAGCTACTATTTTGCATCTCCAGTGTCAAATGTGATGCTGACCTGACCTAATCGGCAGAGACGCAGATCTCCAATGCTCACACTTCACATCCTAAAACGCACTTGCAGTGTACCCTGCTCTCAATTCCCATTGCAATTCCACATCTGGGATCTAAGGGCATGCTTTGTGGGCACTAAAGCGCCGATTTCCTCCTCAATGCGAAGGTTGACCTTACTGAGGAAAGTGCGCATCGTAATCGGCGATGTAGGCTTAGGGATTTCGCCGGGTAACACTCAACGCAATCGTGACGTTCGCTAGATAGCTTGGTTGATATCAGGTGCAAATCCCACAAAATCCTTTGCATGGTCGGGCATTTATGTCTACCAGGGAAGTATCAGTTTTCTTCCAGAATAAAATTTCCACTTTAACAGCAGCTGCTCACGAAATCCTCAGGCATGTGGTGGTGGTGGACGGGGTGAAGAAGAAGGCCCGCCCTCGTCAACACCATCTTCTCCAGTTTGGGGCGACACCACACTCTTCCCTCGGCTCAACAAACGTCGGAAGCTCGCTGAAGCACGCGCCATCGGCGACAAAACATTCGACGAATTGCTGACAGCCGCTGGACCAGACCTCGGAATGTCGATAGTAACCTGAAACGGCGCACGAATACTGGACGCCGCCCTCGCTTCTGCACTCCCTCCTGCACCTGCACTGCTCATCTGCGCATGATTCCCCCAGAATAACACATTGGACGGGATTCCGGACGGTGTCTCGTAATCTTTTATGTTTTGCTGCTGATCAACCGCAACGGCCGTTTCCGACGTACCACTTCGGCGATGATTCTCGCGGCCCTGATCCTCCGCAGTGCGGGGGAAACTATTGCCTCTTGGGGAACTATTCAAGGCCGGCAACTGTGCCGGGCTCCGTTGGCTCCTCCTGGAAGCTCGCATGGCCGGCATGAAGGGCGCTCCTAGGTCACCCATAACGGGCGCTTCCATCTGAGGAGGCTCGCTTATCTGCATCACGGTGGCGGCCTCGGTCTTCTTCTCAGTTTCATCAGCTCCCACACTCGTGCGACACAACGGGCATGTGGAGTGCGAGTGCAACCACATGTCAATACAATCCAAGTGGAAACTATGGTCACACTTCGGCAACGTGCGGCCTTTCTCACGCAACTCAAATTCTTCCAAACAAACCGCGCACTCGAACCCACGCTTTGCCCTCTCACCGTCGAATTCGAAAGTGGGCAGAGCTTCAATAACAGCCCGCTCAAGCCCCACTGCCTGCGTCACCGGAGTAGCGTTCACAGGGACAGTGTAACGGCGTCTTCGCCATGATAACGTACGCAAGGTGCCATCGCTTGCGACGATCTGGTGC Nucleotide sequenceof the partial ZF-3 from Physcomitrella patens (SEQ ID NO:3)GCACCAGCGCTTTTAAACAATCAATATCTGAGCAGGTTGATGGCGAAGATTCAAGATGTATCAAGAAGTGGCGCTAGTGATCAATCTGGGCATGAAAGACCCCTTGACGTTTTACGCAAGGTGAAAGGAAATGATGCTTGTGCCGACTGCGGTGCTGCTGATCCCGATTGGGCTTCGCTGAATCTTGGGATTCTTGTGTGTATTGAGTGCTCAGGAGTACACAGAAACATGAGCGTTCAGATTTCTAAGGTCCGTTCGTTGACGTTAGATGTCAAAGTTTGGGAGCCTTCTGTAATGAGCTATTTTCAATCTGTCGGAAACTCCTACGCTAATTCTATATGGGAAGAGCTTTTGAATCCCAAGTCCTCAGAGGAGTGAAGTGAGAGAAACGTTAATGACGAGGGACAATCGGGCGTTTTAAGTGCTAGCAGAGCAAGGCCAAGACCTAGAGACCCCATACGTATCAAAGAAGATTTATCAATGCAAAGTA TGTGGAGAAAANucleotide sequence of the partial ZF-4 from Physcomitrella patens (SEQID NO:4) GCACGAGCTGCCCCATTCGAGCCCACTCGCACGAGAAGATACGAGCCGCGCTTTGGCCGAGTGGTCGTAAGTAGAAGTAAGGTCCGCGGCCGCTGCGGTCTGTGAAATTCTCTCGGACGGAGAGAAGGGTGTTCTGCTGGTTTCTTCGCACAGAGACTTCTCCTGCACCTTTTCTTCTTCCTCTACATCGTCTCCTGCGACGACTACATTGTGTGGGAGCAGTGGCAAGCTTCCTGGCCACCCCGGGCTTCCTCTCAGTCAGTGGCTCACGTCTCCCAGCTAGGCCTCCCATCGCGTCTTGCCGGCTCAATCGGGTGTCTTGCTCTGTTTTTTAACCTCTCTCCCTTCCGGCCCTCTTATTGCTCTCCAGTCACTTCCGCCGGATCGCGACTTTTGTAGCCATTTGGGGGTTGGGTGTTATAAGTTTGCCCTGAGGGTGTGAGTTGCTTTGTGTGTCTTTTGTAGTAGTACTTTGCTTGTTGGGTGCGGAAGGGAACCTTTGAGAAGTCGACCCATTCTCTAGTTTTGCACCAGTCCCGCTTAGTGTGTGTGTCATTAGTGTTGGTTGCAAGTCTGAAGCCTTGAGCGAGATTTGCAGGATTTTCTCATACGCTTCTGATTAGGAAAGATAGATCCTTATTAGTCTGTTAAAGATGGCCACCGAGCGTGTGTCTCAGGAGACGACCTCGCAGGCCCCTGAGGGTCCAGTTATGTGCAAGAACCTTTGCGGCTTCTTCGGCAGCCAAGCTACCATGGGGTTGTGCTCGAAGTGCTACCGAGAGACAGTCATGCAGCGAAGATGACGGCTTTAGCTGAGCAAGCCACTCAGGCTGCTCAGGGGACATCTGCCAGAGCTGCTGCTGTTCAGCCCCCCGCTCCTGTACATGAGACCAAGCTCACATGCGAGGTTGAGAGAACAATGATTGTGCCGCATGAATCTTCCAGCTATCAACAAGAGGTGGTTACCGCGGCTGCAGCTGCCCCTCAGGCAGTGAAGTGCTCTATCGCAGGTCCCTCTAGACCCGAGCCCAATGGATGCGGATCTTGGAGGAAGCGTGTTGGATTGACAGGATTTAAGTGTGGCTGTGGCAACCTCTACTGCGCTTTACATCGGTACTCGGACAAACACACTTGCACATATGACTACAAAGCCGCAGGGCAGGAAGCGATTGCGAAAGCTAATCCTCTTGTCGTGGCCGAGAAGGTTGTCAAGTTTTGATGAGCATCCGTTAAGCTTTTCTGCCGACGATTTAGGCTTCATACATTGAGTAACTCTACATCTTTCTTCTTTATCGAGAGAGCGAGTCGCATCAAGATGAAGTCGAGGGGTGCGCGTCGGTTTTGGGGAGAGGGGATTTCTTTCCCCTTTCCCCCCTTGGCGGCATCGTGTTTTATGTGTACAGAAGTAGGTTAGGACAAGATAGAATCATATGCCAGATCAATTGATAGTCCTCTTTAAGGAGGACACTTATTACACAATAAAAAATCCTGGGTAATGCATGCCTTGATTGTGTTGTTTTTTCCTCGTGC Nucleotide sequenceof the partial ZF-5 from Physcomitrella patens (SEQ ID NO:5)GCGTGGGGCGTCTACACTAGTTTATCCCCGGGCTGAGGAATTCGGCACCAGATTTGTCAATCAAAAGAAGTTAGTTGCGGGTGATGCTATTGTATTTCTTCGCATCGCATCTGGCGAACTTTGTGTCGGCGTGCGCCGTTCAATGAGGGGTGTCAGCAACGGAGAATCCTCATCTTGGCACTCCTCAATCAGTAATGCTTCAACGATTCGGCCATCTCGATGGGAGGTGAAGGGCACAGAAAGTTTCTCGGACTTTTTAGGTGGCGTTGGTGATAATGGGTACGCACTGAATAGCTCAATTCGGTCTGAAAACCAGGGCTCTCCAACAACGAGTAGCTTTGCACGGGACCGTGCTCGTGTTACTGGGAAGTCCGTTCTAGAAGCTGCTGGACTCGCCGTCTCCGGTGAACGTTTTGAGGTTGTGTATTATGCTGGTGCTAGCACAGCTGAGTTCTGTGTCAAAGCTGGGCTTGTTAAACGTGCGCTAGAGCAATCGTGGTACGCTGGAATGCGCTTCAAAATGGCATTTGAAACTGAAGACTCCTCGAGGATAAGCTGGTTTATGGGAACTATTGCTGCTGTTCAAGCAGCAGATCCAGTAACTTGTGGCGTAGTTCTCGATGGCGGGTCTGCAGGTCACCTTGGGATGAGCGGACCTATTGCAGGAGTGATCGTGTAGCCATGGAGTA Nucleotide sequence of the partialMYB-1 from Physcomitrella patens (SEQ ID NO:6)GCAGCAGTGTTCCCTTTCATATGCTCAGCATGTCCGCCAATGAGCGCGCCTGTTGTGTACAGTCTGTGGAGAGCTGTAGAAAATTCAATTCCGATTTCAAAATATCCAGCGACGATGACACGGAACATGGGAGTTTGGAGGACGACATGAAGGAGTTGAACGAAGACATGGAAATTCCCTTAGGTCGAGATGGCGAGGGTATGCAGTCAAAGCAGTGCCCGCGCGGCCACTGGCGTCCAGCGGAAGACGACAAGCTGCGAGAACTAGTGTCCCAGTTTGGACCTCAAAACTGGAATCTCATAGCAGAGAAACTTCAGGGTCGATCAGGGAAAAGCTGCAGGCTACGGTGGTTCAATCAGCTGGACCCTCGCATCAACCGGCACCCATTCTCGGAAGAAGAGGAAGAGCGGGTGCTTATAGCACACAAGCGCTACGGCAACAAGTGGGCATTGATCGCGCGCCTCTTTCCGGGCCGCACAGACAACGCGGTGAAGAATCACTGGCCC Nucleotide sequence of the partial CABF-3 fromPhyscomitrella patens (SEQ ID NO:7)GCACCAGGTCTTCGACTTTGCTTCAGCACGCGCGCGTTGTGGTCGATCTCTCGCTGGAGCAACAGGTTGTCTTGTCGCTGCCATTGCTAAAGCCATTCTTACTTCTAGCACTTCTCGGAGGTTATTGATTTCTCGCAAATTGCTGTTCCACCTGCCCTCTTTCGTGAGGGAGTTCGAAGCTGAAAAGTAATGAGCTGAAGATTAAGGTCTTTTACGAGTGAACAGCGAGCACACAGCTAGCAACTCTTTCGGAGAATACTCCAGGCGAAATTGGTCGGATGGCCGATAGCTACGGTCACAACGCAGGTTCACCAGAGAGCAGCCCGCATTCTGATAACGAGTCCGGGGGTCATTACCGAGACCAGGATGCTTCTGTACGGGAACAGGATCGGTTCCTGCCCATCGCGAACGTGAGCCGAATCATGAAGAAGGCGTTGCCGTCTAATGCAAAAATTTCGAAGGACGCGAAAGAGACTGTGCAGGAGTGTGTGTCCGAGTTCATCAGCTTCATCACTGGTGAGGCGTCAGATAAGTGC Nucleotide sequence ofthe partial SFL-1 from Physcomitrella patens (SEQ ID NO:8)GCACGAGTTTTCTTGTGTCAAAGCAGCAGAAGAAATCCACTTCTGGTAGTATTCAAACATAAAAGAATGGAAACTTATGTAACAGTCTACTTTCTGATCGAAACATTACCAAATGCCTTTTTCCTGGTTTGGTAGGTACTATCAATCAGCAGCAATTAAATAGCGTCAGATTTCACATCTAAGTACTCTCGTAGAATGCTGTTCCGGCTGGGTTGCCTCAGCTTGCGCATCGCTTTTGCCTCAATTTGTCTTATCCTTTCCCGAGTAACTTTAAAGATTTGACCTATTTGTTCTAAAGTGTTGGACCGCCCATCGTCCAATCCAAAACGCAGTCTTAGCACCTCCGTCTCTCTTGGGTTCAATGTGCGTAGAACGCCCTCTATATCTTGTCGCATCAATTGCTTTACGATTGCGTCCTCAGGTGAATCCACATCTGTGTCTGCGACAAGTTCCCCAAGTGTANTGTCCCCATCTTTGCCAATGGGCCGCTCCATCGAACCTGGTGCCTTTGCTGATTTCACTACAGATTTCAGTTTCTCAACAGTCAAGCCCACTAGCTCAGCCACTTCCTCGTTACGTGCTTCCCGCCCATGCTCCTG Nucleotide sequence of thefull-length APS-2 from Physcomitrella patens (SEQ ID NO:9)GCGATATCGGAAGAAGAACCAAGGGAATGCGGTTAGCGGCGAAAGACACTAGCGGTCGGAATGCCTTTAAATTTCGGAATATTGATTTAAATAAGGCCCCATCGGCATGGGATACCGAAGAAGTTTCTGCTAGCAACACTGGCGATACGACCAGTTTTAGGGGGGTTGGGCACCGGCCCGAGCTAAACAAATGGGTGACAGAAATTCGACCCACATCTCAGAAACGTAAGATATGGCTGGGAACATATGAAACTCCGGAAGAAGCTGCCCGGGCCTACGATGTTGGCATCTTCTACAGAAAGAAGAAGATCCCGTACAATTTTGAGGATTCGCCAGAGCAGCTGCAGCTATATCCCATCCCCCCGGAACTGCCTTGGGAGAGTTTTGCCGCCCTCGTGAAGCAGAGGGCTACTTCCGCGGCGAAGAGGGCGAGGGTGCCTTCCTCAAGCTAGAGCGATACGCAGGGTAATTGACGCTGGTCGGCTGAGAGAATGATCATCAAGGTGGGTTGTGATGTTCTATGCTCGGATGAGTGGCTTGAAGGGTTCTGGTTCCAACCATGAGAGCATGACGCGAGTGCCACACGGATGGAGCTTGTGAATGGAGTGGTAGACTGTAGATGGTTTTTGTAACGGCTTGAGTAATAACGGAAGCTTCATGGCTTGAATGACCAGCCATGGTGGTGTGCAAGTGAAGATCGCTGCTTGTGTGAAGGTTTCCATCTTTCCCATGCCCGTCTTCCACTTTGCTACACGTTGCTAGTGTCACTTGAAGAATTGATTCATGGACCCTGCTCTGCTTTCCCCTGTTACGAAGTTCTTATGGTAGAGTTCACCGAACGCAAGCTGTCTAGGAAGTTGACAGTTTGTGGGAGCCAAAAACTCTACTTGAGCTACTGTGTGCACGGCTTCTGAGTCCTCCAGCGAGGAGCCTGTATATTATTGGATGGTGCAGGATGGGTCGCTTGGTGCCTTTCTCTTTTTCCTTTTCCTCTTTTTGTAAATGGTTTTCCTTCTATGAATATGTGAAGCTCCTCCCACGGAAGCATAGAGCTCGC Nucleotide sequence of the full-length ZF-2 fromPhyscomitrella patens (SEQ ID NO:10)ATCCCGGGATCAGGAAGCTGTCAAGGAAGAGATGGAAATCTTGCTCCATACAATTACTACGGGCCGCCACCGGGCAGTAACAATTATGTCGTGAACAGCAAGATTATGGTCGTGGGTGTCGCGGTTCTCTTCGCTGTCGTCGTCTTCATCCTCTGCCTCCACATCTACGCCAAGTGGTTCTGGCGCAATCAAGGTGCCATGGTCGCAAGCGATGGCACCTTGCGTACGTTATCATGGCGAAGACGCCGTTACACTGTCCCTGTGAACGCTACTCCGGTGACGCAGGCAGTGGGGCTTGAGCGGGGTGTTATTGAAGCTCTGCCCACTTTCGAATTCGACGGTGAGAGGGCAAAGCGTGTGTTCGAGTGCGCGGTTTGTTTGGAAGAATTTGAGTTGGGTGAGAAAGGCCGCACGTTGCCGAAGTGTGACCATAGTTTCCACTTGGATTGTATTGACATGTGGTTGCACTCGCACTCGACATGCCCGTTGTGTCGCACGAGTGTGGGAGCTGATGAAACTGAGAAGAAGACCGAGGCCGCCACCGTGATGCAGATAAGCGAGCGTCCTCAGATGGAAGCGCCCGTTATGGGTGACGTAGGAGCGCCGTTGATGGCGGGCATGCGAGCTTCCAGGAGGAGCCAACGGAGCCGGGGACAGTTGCCGGCGTTGAATAGTTCCCCAAGAGGCAATAGTTTGCCCCGCACTGCGGAGGATCAGGGCGGGGAGAATCATCGCCGAAGTGGTACGTCGGAAACGGCCGTTGCGGTTGATCAGCAGCAAAACATAAAAGATTACGAGACACCGTCCGGAATCCCGTCCAATGTGTTATTCTGGGGGAATGATGCGCAGATGAGCAGTGCAGGTGCAGGAGGGAGTGCAGAAGCGAGGGCGGCGTCCAGTATTCGTGCGCCGTTTGAGGTTACTATCGACATTCCGAGGTCTGGTCCAGCGGCTGTCAGCAATTCGTCGAATGTTTTGTCGCCGATGGCGCGTGCTTCAGCGAGCTTCCGACGTTTGTTGAGCCGAGGGAAGAGTGTGGTGTCGCCCCAAACTGGAGAAGATGGTGTTGACGAGGGCGGGCGTTCTTCTTGACGCCGTCCACCACCACCACATGCCTGAGGATTTCGTGAGGAGGTGCTGTTAAAGTGGAAATTTTATTCTGGAAGAAAACTGATACTTCCCTGGTAGACATAAATGCCCGACGATGCAAAGGATTTTGTGGGATTTGCACCTGATATCAACCAAGCTATCTAGCGAACGTCACGATTGCGTTGAGTGTTACCCGGCGAAATCCCTAAGCCTACATCGCCGATTACGATGCGCACTTTCCTCAGTAAGGTCAACCTTCGCATTGAGGAGGAAATCGGCGCTTTAGTGGCCACAAAGCATGCCCTTAGATCCCAGATGTGGAATTGCAATGGGAATTGAGAGCAGGGTACACTGGAAGTGCGTTTTAGGATGTGAAGTGTGAGCATTGGAGATCTGCGTGTCTGCCGATTAGGTCAGGTCAGCATCACATTTGACACTGGAGATGCAAAATAGTAGCTATTCCTTCTTCACTTCTTCCGATGGTGTACCGTAGTGTAATATATAGCGCTAGATGTGCAATAAGACGCCACAGTCTGAGGCTCAAGATGTAATATAGATCATGCACGAAGCAGTTAAGCATCTCTAGGTTAGGAGATTGAATGCCACAGTGCTTCAGTAACATGTAGAGTTGTAAATAAACGGGAAATCGCCTTCTGCAAGGCATCTGACTGACACGAACCTAGTGAACATATCAAGGTAATTGCCATTTTACGATAACCAGCTTATTGCAAGGCAAGCGCCAGAGCTCGC Nucleotide sequence of thefull-length ZF-3 from Physcomitrella patens (SEQ ID NO:11)ATCCCGGGAGGAGGACTTGCGGAATGCAAAATCACAATTTGAGCAGGCTCGATTCAATTTGATGAGAGCACTTACCAATAGTGAGGCAAAAAAGAAGTTCGAGTTCCTTGAAGCCGTGAGTGGTACAATGGATGCACATCTCAGGTACTTTCAAGCAGGGCTATGAGTTGCTACATCAAATGGAACCTTACATCCATCAGGTGTTAACATATGCTCAACAGTCCAGAGAAAGGGCCAACTACGAGCAAGCAGCACTTGCAGATCGTATGCAGGAGTACAGGCAGGAAGTTGAGAGAGAGAGCCAAAGGTCGATTGATTTTGACAGCTCTTCTGGAGATGGTATTCAAGGTGTTGGCCGCAGTTCACATAAGATGATTGAGGCAGTCATGCAATCGACCCCAAAAGGGCAGATCCAGACTCTTAAGCAGGGATACCTGTTAAAGCGTTCAACAAATTTGCGAGGTGACTGGAAGCGGAGGTTTTTTGTGTTGGATAGCAGAGGAATGCTGTATTATTATCGGAAACAGTGGGGCAAGCCTACAGACGAGAAAAATGTAGCACATCACACTGTGAATCTGCTGACGTCTACAATCAAGATAGACGCAGAACAATCAGATCTTCGTTTCTGCTTTCGGATTATTTCTCCAGCTAAAAGTTATACCCTGCAGGCAGAAAATGCCATTGACAGAATGGATTGGATGGACAAAATTACAGGGGTGATTTCGTCGCTTTTAAACAATCAAATATCTGAGCAGGTTGATGGCGAAGATTCAGATGTATCAAGAAGTGGCGCTAGTGATCAATCTGGGCATGAAAGACCCCTTGACGTTTTACGCAAGGTGAAAGGAAATGATGCTTGTGGGGACTGCGGTGCTGCTGATCCCGATTGGGCTTCGCTGAATCTTGGGATTCTTCTGTGTATTGAGTGCTCAGGAGTACACAGAAACATGAGGGTTCAGATTTCTAAGGTCCGTTCGTTGACGTTAGATGTCAAAGTTTGGGAGCCTTCTGTAATGAGCTATTTTGAATCTGTCGGAAACTCCTACGCTAATTCTATATGGGAAGAGCTTTTGAATCCCAAGTCCTCAGAGGAGTCAAGTGAGAGAAACGTTAATGACGAGGGACAATCGGGCGTTTTAAGTGCTAGCAGAGGAAGGCCAAGACCTAGAGACCCGATACGTATCAAAGAAAGATTTATCAATGGAAAGTATGTGGAGAAAAAATTTGTCCAAAAGTTGAAGGTGGATTCTCGAGGCCCGTCAGTGACACGGCAGATCTGGGATGCTGTCCAGAAGAAAAAAGTGCAGCTTGCTCTTCGTCTTCTTATCACTGCTGATGCTAACGCCAACACAACCTTCGAGCAAGTAATGGGTGGTACCGAGTCTTCGTGGTCGTCTCCACTTGCAAGCCTCGCTGGAGCTCTCTTACGAAAGAACTCTCTCAGTGCCTCTCAGAGTGGTCGCAGGAACTGGAGCGTACCTTCACTATTGTCTTCTCCAGACGATCCGGGGTCCCGTTCAGGAGCTTTAAGGCCTGTTTCGAGAAGTCCTGATGCAGCAGGCAGCGGAGGGATTGATGAGAAAGATTTGCGGGGCTGCAGTTTGCTCCATGTTGCCTGCCAAATCGGAGATATTAGCCTGATCGAGCTGGTACTTCAATACGGGGCGCAAATCAATTGTGTGGATACCCTGGGTCGAACTCCTCTTCATCACTGTGTCTTGTGCGGCAACAATTCGTGTGCAAAGCTCCTGCTCACAAGAGGGGCGAAGGCGGGTGCCGTAGACAAAGAGGGAAAAAGTCGGCTGGAGTGTGCAGTGGAGAAGCTAGGTGCTATCACGGATGAAGAATTGTTCATAATGCTTTCTGAAACCAGTAGATGACAGCACATTTGTGCCTGAGTTGCTTTGTGTATAAATCTCAACATCAACTTGTTTGCTAGCACCTGTAAGGCTAGTTTGTTTGGGTAGTTTGCATTCTTGTTCTACCGTTTTATCTTCCCATTACGTGAGCATAAGAGAGAGTGGAAGCAGGTGGATATCGC Nucleotide sequence of the full-length ZF-4from Physcomitrella patens (SEQ ID NO:12)ATCCCGGGCACCAGTCCCGCTTAGTGTGTGTGTCATTAGTGTTGGTTGCAAGTCTGAAGCCTTGAGCGAGATTTGCAGGATTTTCTCATACGCTTCTGATTAGGAAAGATACACCCTTATTAGTCTGTTAAAGATGGCCACCGAGCGTGTGTCTCAGGAGACGACCTCGCAGGCCCCTGAGGGTCCAGTTATGTGCAAGAACCTTTGCGGCTTCTTCGGCAGCCAAGCTACCATGGGGTTGTGCTCGAAGTGCTACCGAGAGACAGTCATGCAAGCGAAGATGACGGCTTTAGCTGAGCAAGCCACTCAGGCTGCTGAGGCGACATGTGCCACAGCTGCTGCTGTTCAGCCCCCCGCTCCTGTACATGAGACCAAGCTCACATGCGAGGTTGAGAGAACAATGATTGTGCCGCATCAATCTTCCAGCTATCAACAAGACCTGGTTACCCCCGCTGCAGCTGCCCCTCAGGCAGTGAAGTCCTCTATCGCAGCTCCCTCTAGACCCGAGCCCAATCGATGCGGATCTTGCAGGAAGCGTGTTGGATTGACAGGATTTAAGTGTCGCTGTGGCAACCTCTACTGCGCTTTACATCGGTACTCGGACAAACACACTTGCACATATGACTACAAAGCCGCAGGGCAGGAAGCGATTGCGAAAGCTAATCCTCTTGTCGTGGCCGAGAAGGTTGTGAAGTTTTGATGAGCATCCGTTAAGCTTTTCTGCCGACGATTTAGGCTTGATACATTGAGTAACTCTACATCTTTCTTCTTTATCGAGAGAGCGAGTCGCATCAAGAGCTCGCC Nucleotide sequence of thefull-length ZF-5 from Physcomitrella patens (SEQ ID NO:13)ATCCCGGGTATCGATCTGGAGCCCGTTGCAAACTGAATGGTGTATTTTATAGGGCAAAAGTCTGATCTATATGGAATGCATCCTCTGAGAGTTGCAAATGATGGACTGCATGTCACTCTGGGTTATTCTCGATCACCTAGCTTTGCTGGAGTTCAAATTGGTGAGTACGAGTATTATGAGTGATCTCGAGTTTATGGTCCCCTTCTTTCATGATCAAGGGTAATTTATATGAAGGGTGTATATGAGAGATACGCACTTATTGAGTGGACCTTTTCTCATACTGCATTTACACCCCTGTCAGTTGCAGCATCCTGGTTTGGAATGCCGGGTCCAGTCCCTCTATTATCCATGAGTGTAAAATCGGAGAGTCTCGATGACATTGGAGGTCACGAGAAAAAATCTGTAACTGGGTCGGAAGTGGGTGGCCTCGATGCTCAGCTGTGGCATGCCTGTGCTGGGGGTATGGTTCAACTGCCTCATGTGGGTGCTAAGGTTGTCTATTTTCCCCAAGGCCATGGCGAACAAGGTGCTTGAACTCCCGAGTTCCCCCGCACTTTGGTTCCAAATGGAAGTGTTCCCTGCCGAGTTGTGTCAGTTAACTTTCTGGCTGATACAGAAACAGACGAGGTATTTGCTCGTATTTGCCTGCAGCCTGAGATTGGCTCCTCCGGTCAGGATTTAACAGATGATTCTCTTGCGTCTCCGCCTCTAGAGAAACCAGCTTCATTTGCCAAAACGCTCACTCAAAGTGATGCAAACAACGGTGGAGGCTTTTCAATACCTCGTTATTGTGCTGAAAGTATTTTCCCACCTCTCGATTACTGTATCGATCCTCCTGTTCAAACTGTTCTTGCAAAGATGTCCATGGAGAGGTGTGGAAATTTCGTCACATTTACAGGGGGACTCCACGTCGACATTTGTTACCACAGGATGGAGCACATTTGTCAATGAAAAGAAGTTAGTTGCGGGTGATGCTATTGTATTTCTTCGCATCGCATCTGGCGAACTTTGTGTCGGCGTGCGCCGTTCAATGAGGGGTGTCAGCAACGGAGAATCCTCATCTTGGCACTCCTCAATCAGTAATGCTTCAACGATTCGGCCATCTCGATGGGAGGTGAAGGGCACAGAAAGTTTCTCGGACTTTTTAGGTGGCGTTGGTGATAATGGGTAGGCACTGAATAGCTCAATTCGGTCTGAAAAGCAGGGCTCTCCAACAACGAGTAGCTTTGCACGGGACCGTGCTCGTGTTACTGCGAAGTCCGTTCTAGAAGCTGCTGCACTCGCCGTCTCCGGTGAACGTTTTGAGGTTGTGTATTATCCTCGTGCTAGCACAGCTGAGTTCTGTGTCAAAGCTGGGCTTGTTAAACGTGCGCTAGAGCAATCGTGGTACGCTGGAATGCGCTTCAAAATGGCATTTGAAACTGAAGACTCCTCGAGGATAAGCTGGTTTATGGGAACTATTGCTGCTGTTCAAGCAGCAGATGCAGTACTTTGGCCTAGTTCTCCATGGCGGGTTCTGCAGGTCACTTGGGATGAGCCGGACCTATTGCAGGGAGTGAATCGTGTAAGCCCATGGCAGTTAGAGCTTGTGGCGACACTTCCTATGCAGCTGCCCCCTGTCTCTCTCCCAAAAAGAAACTGCGCACTGTCCAGCCTCAAGAGCTTCCACTTCAGCCCCCTGGACTGCTAAGCCTGCCGTTGGCAGGGAGTAGCAACTTTGGTGGGCACTTGGCCACCCCCTGGGGGAGCTCTGTTCTTTTGGATGACGCCTCTGTTGGCATGCAGGGGGCCAGGCATGATCAATTCAACGGGCTTCCAACTGTGGATTTCCGAAATAGTAACTACAAACATCCTCGGGAGTTTTCTAGGGACAATCAGTACCAGATTCAAGATCATCAAGTCTTCCATCCTAGACCTGTATTAAATGAGCCCCCTGCGAGAACACTGGCAAGTACTTCTCTCTTTTACCTAGTCTCCAGCGACGGCCAGATATCTCTCCTAGTATTCAGCCCTTAGCCTTCATGTCTGCTTCTGGAAGCTCACAGCTGGAGACTTCTTCAACTAAGACAGCGGCCACCTCTTTTTTCCTATTTGGCCAATTCATTGACCCTTCTTGCACCTCCAAACCTCAGCAGCGTTCCACAGTTATTAATAACGCTTCCGTTGCTGGGGATGGTAAGCATCCTGGCACTAATAACTCATCCTCGGATAACAAATCAGAGGACAAGGACAATTGTAGGGATGTTCAACCCATTCTGAATGGGATTGCTGTAAGATCTGGATTTCGAGCAGATATAGCCGCGAAGAAGTTTCAAGAGAGCGACTCTGCACATCGCACGGAAGCATCACGTGGAAGCCAAGTTAGCAGCTTACCGTGGTGGCAAACACAGGACGCTCACAAGGATCAGGAATTCCATGGAGACAGTCAGAGGCCTCATACTCGTGCATCTGGTAGCCCTGAGGCTAAAGCTTGATCATAGCTCATAACCCTCTCACAGGACGTAATGGGGGTGACAACATGCTAACAGAATTGCACGGTAAAGGAAAACTGTACTAGGCATGTTATATGGGAATTCGGATCGCTTCTTGCAATTAAACACGCTAGCGCCGTTTGGTGCCAATGTTATTCTGGCATTTGTTTTGTTTCCTTTGGAAACAAATTGCTATATTTCAAAGTCCTTTGGAGGAGCTCGC Nucleotide sequence of the full-length MYB-1 fromPhyscomitrella patens (SEQ ID NO:14)ATCCCGGGCTGTTGTGTACAGTCTGTGGAGAGCTGTAGAAAATTCAATTCCGATTTCAAAATATCCAGCGACGATGACACGGAACATGGGAGTTTGGAGGACGACATGAAGGAGTTGAAGGAAGACATGGAAATTCCCTTAGGTCGAGATGGCGAGGGTATGCAGTCAAAGCAGTGCCCGCGCGGGCACTGGCGTCCAGCGGAAGACGACAAGTTGCGAGAAGTAGTGTCCCAGTTTGGACCTCAAAACTGGAATCTCATAGCAGAGAAACTTCAGGGTCGATCAGGGAAAAGCTGCAGGCTACGGTGGTTCAATCAGCTGGACCCTCGCATCAACCGGCACCCATTCTCGGAAGAAGAGGAAGAGCGGCTGCTTATAGCACACAAGCGCTACGGCAACAAGTGGGCATTGATCGCGCGCCTCTTTCCGGGCCGCACAGACAACGCGGTGAAGAATCACTGGCACGTTGTGACGGCAAGACAGTCCCGTGAACGGACACGAACTTACGGCCGTATCAAAGGTGCGGTACATCGAAGAGGCAAGGGTAACCGTATCAATACCTCCGCACTTGGAAATTACCATCACGATTCGAAGGGAGCTCTCACAGCCTGGATTGAGTCGAAGTATGCGACAGTCGAGCAGTCTGCGGAAGGGCTCGCTAGGTCTGCTTGTACCGGCAGAGGCTCTCCTGCTCTACCCACCGGTTTCAGTATACCGCAGATTTCCGGCGGCGCCTTCCATCGACCGACAAACATGAGTACTAGTCCTCTTAGCGATGTGACTATCGAGTCGCCAAAGTTTAGCAACTCCGAAAATGCGGAAATAATAACCGCGCCCGTGCTGCAAAAGCCAATGGGAGATCCCAGGTCAGTATGCTTGCCGAATTCGACTGTTTCCGACAAGCAGCAAGTGCTGCAGAGTAATTCCATCGACGGTCAGATCTCCTCCGGGCTCCAGACAAGCGCAATAGTAGCGCATGATGAGAAATCGGGCGTCATTTCAATGAATCATCAAGCACCGGATATGTCCTGTGTTGGATTGAAGTCAAATTTTCAGGGGAGTCTCCATCCTGGCGCTGTTAGATCTTCTTGGAATCAATCCCTTCCCCACTGTTTTGGCCACAGTAACAAGTTGGTGGAGGAGTGCAGGAGTTCTACAGGCGCATGCACTGAACGCTCTGAGATTCTGCAAGAACAGCATTCTAGCCTTCAGTTTAAATGCAGCACTGCGTACAATACTGGAAGATATCAACATGAAAACCTTTGTGGGCCAGCATTCTCGCAACAAGACACAGCGAACGAGGTTGCGAATTTTTCTACGTTGGCATTCTCCGGCCTAGTGAAGCATCGGCAAGAGAGGTTGTGCAAAGATAGTGGATCTGCTGTGAAGCTGGGACTATCATGGGTTACATCCGATAGCACTCTTGACTTGAGTGTTGCCAAAATGTCAGCATCGCAGCCAGAGCAGTCTGCGCCGGTTGCATTCATTGATTTTCTAGGCGTGGGAGCGGCCTGAAGGCTGCGGAAAGATTTTAGCAAAGCTTTTATAACGTTTTTTTTGCACAGGGGTGTTTTAGCTTGTATACCAGTAGGCACTTCTACTTCTTTTTCTTCTTTTCTTTTTCCCCTTTTCTTCTCCCCCCACTTTCACCATTTCCGCCATAGCAGCCTTTGAATCACGTAATGGAACCTTTGGCGGCCTGTATGAGGCAGTTTTGGAGGCATCCCTGGACGAAGAATGGATCAAACCGTACTGCGGATGTCATGCTTTGAAGCTGCAATCCGAATTCAGTAGCATGCTGTGGATGACTCAAAAGGAGTAGTGCTTTGTGAAACTAATACTATACAGCGGATTTTGAAGACCCAAGTTTCATGTGGACAAGTCTGAAAAACTTATACGCCACCTCCATGGGCTTCTACGATGAATATGCGCTTTCGGCTTACACTGCGGCTCTTTTTTGCATATATATATACTTCCATTCAATTTTATTTGGAAATGTTTTGAATCTACCTTCTCGTACAAAACTGGGATCAGAAATCTTCCAGGTTGTGGGTCGCAAGTTAACTCTGCAGATTGTGGCTGACACTTGGGCAATGGGCAACTTTATCTTTTTGTTTTTTACGCTTGAACGGACCTCAGGTGTACAGACAGTGATCATGTACATTCGATGCCATCTCTTGGCTTTCATGGAAGTTCAGATATCGGAAACTGTGACAGAGACAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGATTCTTGATGCACTGTGCGCCGAGTTTGAGACTAGTTTAGAAAGATTGATGAAGCTAGCAGTAAATTGTTGGCCTCATCTGAAAGGTACGGCCTTTACTCCGTGAGCCCGGGAT Nucleotide sequence of the full-length CABF-3 fromPhyscomitrella patens (SEQ ID NO:15)ATCCCGGGCAGCGAGCACACAGCTAGCAACTCTTTCGGAGAATACTCCAGGCGAAATTGGTCGGATGGCCGATAGCTACGGTCACAACGCAGGTTCACCAGAGAGCAGCCCGCATTCTGATAACGAGTCCGGGGGTCATTACCGAGACCAGGATGCTTCTGTACGGGAACAGGATCGGTTCCTGCCCATCGCGAACGTGAGCCGAATCATGAAGAAGGCGTTGCCGTCTAATGCAAAAATTTCGAAGGACGCGAAAGAGACTGTGCAGGAGTGTGTGTCCGAGTTCATCAGCTTCATCACTGGTGAGGCGTCAGATAAGTGCCAGAGGGAGAAGAGAAAGACGATCAACGGTGACGACTTGCTGTGGGCCATGAGTACACTTGGTTTCGAAGATTACGTGGAGCCTCTGAAGGTTTACCTACACAAATACCGGGAGCTAGAGGGAGAGAAGGCTTCCACGGCCAAGGGTGGTGACCAGCAAGGAGGGAAAGAAGGGAGTCAAGGTGTTATGGGGTCCATGGGTATGTCGGGCGGAATGAACGGTATGAACGGTACGATGAACGGGAATATGCATGGACATGGAATCCCGGTGTCGATGCAGATGCTGCAGCAGTCGTACGGACAGGAGGCACCTCCAGGGATGATGTATTCCCCTCATCAGATGATGCCGGAATACCAGATGCCAATGCAGTCTGGTGGAAACCAGCCTCGTGGAGTGTAGGAGGTTCCACGGCGAGGAGAATTTGAAATTGGGGAGATTGTCAAGGGCGTGAGGGAGTGAGCTCGC Nucleotide sequence of the full-lengthSFL-1- from Physcomitrella patens (SEQ ID NO:16)ATCCCGGGCTCGGAAGGACTGTGCATTGTCGAGCGCTGAAGGTGGATGATGCTTTGGTGAGCGAGAGCGGTCTTATCAGTGAAGAAGGAGTTTCTCGTGCTGCAGCTGAGGAGGCGATGACGTTAGCTTTAGCAGCTGCAAAGGCCGCCATGGAGGCTGCCTCGTACGCTGATGCGATGCCGTGGAACAGGAGGAGTTTCCGACGGAATTTGATCTGCTGAGACTAGAGAGGGCCAGGTTGAGCGATGTTGAGCATTCTTTTCGGGTTGAATTGGATACAGAGGCTGGCATGATGGAGGCCGAGCAGAGTTATGTGCAGAAGCTAGAATCGTTGTTGGGAGGTGTTTCCACGCTCGTCCGTGAGGAAGAGGAAACTGCATCCGTTTCAGAAGATGAAGATGATTCAAACAGCTTACCTCAAATTCAAGTAGCCGTTAAATCGAAGCGGAAGGGAGAGAGGAGGAAGAGGCGGGAGCGAGCGTTGGAAAGGGCAGAGAAGGTTGCCACCGATCTTGCATCAGCACCCCCTCTCCCAAAACCTAAGAAACCACAGCTTGCGGCGGATCCTTCAGACCCAGTCCGTGCATATTTGCGAGACATAGGAAGGACGAAGTTGCTAACAGCAAGAGAAGAAGTCGATCTCTCTCATCAAATTCAGGATGTTTTGAAGTTGGAGAATATCAAGTCTAACCTTGAGCGAGAGATAGGAAGGAATGCCACAATTGGAGAGTGGAGTAGAGCGGTAGGAATGGAACAGAATGCATTTGAAGCGCGGCTTAAGAAGGGTCGATTCGCCAAGGACAAAATGGTGAATTCGAATTTGCGGTTGGTTGTCTCGATTGCGAAAAACTACCAGGGCCGAGGCATGACTCTTGAAGATTTAATTCAGGAAGGGAGCATGGGATTGGTGAGAGGAGCGGAGAAGTTCGACCCGACCAAGGGGTTTAAGTTCAGCACTTACGGACATTGGTGGATTAGGCAGGCTGTAACGCGATCAATTGCGGATCAATCTAGGACTTTTCGTTTACCTATTCATTTATACGAAGTTATCTCACGTATCAACAAAGCAAAGCGAATGCTGGTTCAGGAGCATGGGCGGGAAGCACGTAACGAGGAAGTGGCTGAGCTAGTGGGCTTGACTGTTGAGAAACTGAAATCTGTAGTGAAATCAGCAAAGGGACCAGGTTCGATGGAGCGGCCCATTGGCAAAGATGGGGACACTACACTTGGGGAACTTGTCGCAGACACAGATGTGGATTCACGTGAGGACGCAATCGTAAAGCAATTGATGCGACAAGATATAGAGGGCGTTCTACGCACATTGAACCCAAGAGAGAGGGAGGTGCTAAGACTGCGTTTTGGATTGGACGATGGGCGGTCCAAGACTTTAGAAGAAATAGGTCAAATCTTTAAAGGTACTCGGGAAAGGATAAGACAAATTGAGGCAAAAGCGATGCGCAAGCTGAGGCAACCCAGCCGGAACAGCATTCTACGAGAGTACTTAGATGTGAAATCTGACGGTATTTAATTGGTGCTGATTGATAGTACCTACCAAACCAGGAAAAAGGCATTTGGTAATGTTTCGATCAGAAAGTAGACTGTTACATAAGTTTCCATTCTTTTATGTTTGAATACTACCAGAAGTGGATTTCTTCTGCTGCGAGCTCGC Deduced amino acidsequence of APS-2 from Physcomitrella patens (SEQ ID NO:17)MRLAAKDTSGRNAFKFRNIDLNKAPSAWDTEEVSASNTGDTTSFRGVRHRPELNKWVTEIRPTSQKRKIWLGTYETPEEAARAYDVGIFYTKKKIPYNFEDSPQQLQLYPIPPELPWESFAALVKQRATSAAKRARVPSSS* Deduced amino acid sequence of ZF-2 fromPhyscomitrella patens (SEQ ID NO:18)MVVAVAVLFAVVLFILCLHIYAKWFWRNQGAIVASDGTLRTLSWRRRRYTVPVNATPVTQAVGLERAVIEALPTFEFDGERAKRVFECAVCLEEFELGEKGRTLPKCDHSFHLDCIDMWLHSHSTCPLCRTSVGADETEKKTEAATVMQISEPPQMEAPVMGDVGAPFMAAMRASRRSQRSRGQLPALNSSPRGNSLPRTAEDQGGENHRRSGTSETAVAVDQQQNIKDYETPSGIPSNVLFWGNHAQMSSAGAGGSAEARAASSIRAPFQVTIDIPRSGPAAVSNSSNVLSPMARASASFRRLLSRGKSVVSPQTGEDGVDEGGPSSSPRPPPPHA* Deduced aminoacid sequence of ZF-3 from Physcomitrella patens (SEQ ID NO:19)MTALTNSEAKKKFEFLEAVSGTMDAHLRYFKQGYELLHQMEPYIHQVLTYAQQSRERANYEQAALADRMQEYRQEVERESQRSIDFDSSSGDGIQGVGRSSHKMIEAVMQSTPKGQIQTLKQGYLLKRSTNLRGDWKRRFFVLDSRGMLYYYRKQWGKPTDEKNVAHHTVNLLTSTIKIDAEQSDLRFCFRIISPAKSYTLQAENAIDRMDWMDKITGVISSLLNNQISEQVDGEDSDVSRSGASDQSGHERPLDVLRKVKGNDACADCGAADPDWASLNLGILLCIECSGVHRNMSVQISKVRSLTLDVKVWEPSVMSYFQSVGNSYANSIWEELLNPKSSEESSERNVNDEGQSGVLSASRARPRPRDPIPIKERFINAKYVEKKFVQKLKVDSRGPSVTRQIWDAVQNKKVQLALRLLITADANANTTFEQVMGGTESSWSSPLASLAGALLRKNSLSASQSGRRNWSVPSLLSSPDDPGSRSGALSPVSRSPDAAGSGGIDEKDLRGCSLLHVACQIGDISLIELLLQYGAQINCVDTLGRTPLHHCVLCGNNSCAKLLLTRGAKAGAVDKEGKTPLECAVEKLGAITDEELFIMLSETSR* Deduced amino acid sequence ofZF-4 from Physcomitrella patens (SEQ ID NO:20)MATERVSQETTSQAPEGPVMCKNLCGFFGSQATMGLCSKCYRETVMQAKMTALAEQATQAAQATSATAAAVQPPAPVHETKLTCEVERTMIVPHQSSSYQQDLVTPAAAAPQAVKSSIAAPSRPEPNRCGSCRKRVGLTGFKCRCGNLYCALHRYSDKHTCTYDYKAAGQEAIAKANPLVVAEKVVKF* Deduced amino acid sequence of ZF-5 fromPhyscomitrella patens (SEQ ID NO:21)MPGPVPLLSMSVKSESLDDIGGHEKKSVTGSEVGGLDAQLWHACAGGMVQLPHVGAKVVYFPQGHGEQAASTPEFPRTLVPNGSVPCRVVSVNFLADTETDEVFARICLQPEIGSSAQDLTDDSLASPPLEKPASFAKTLTQSDANNGGGFSIPRYCAETIFPPLDYCIDPPVQTVLAKDVHGEVWKFRHIYRGTPRRHLLTTGWSTFVNQKKLVAGDAIVFLRIASGELCVGVRRSMRGVSNGESSSWHSSISNASTIRPSRWEVKGTESFSDFLGGVGDNGYALNSSIRSENQGSPTTSSFARDRARVTAKSVLEAAALAVSGERFEVVYYPRASTAEFCVKAGLVKRALEQSWYAGMRFKMAFETEDSSRISWFMGTIAAVQAADPVLWPSSPWRVLQVTWDEPDLLQGVNRVSPWQLELVATLPMQLPPVSLPKKKLRTVQPQELPLQPPGLLSLPLAGTSNFGGHLATPWGSSVLLDDASVGMQGARHDQFNGLPTVDFRNSNYKHPREFSRDNQYQIQDHQVFHPRPVLNEPPATNTGNYFSLLPSLQRRPDISPSIQPLAFMSASGSSQLETSSTKTAATSFFLFGQFIDPSCTSKPQQRSTVINNASVAGDGKHPGTNNSSSDNKSEDKDNCRDVQPILNGIAVRSGFRADIAAKKFQQSDSAHPTEASRGSQVSSLPWWQTQDAHKDQEFHGDSQTPHTPASGSQ* Deduced amino acid sequence of MYB-1 fromPhyscomitrella patens (SEQ ID NO:22)MKELNEDMEIPLGRDGEGMQSKQCPRGHWRPAEDDKLRELVSQFGPQNWNLIAEKLQGRSGKSCRLRWFNQLDPRINRHPFSEEEEERLLIAHKRYGNKWALIARLFPGRTDNAVKNHWHVVTARQSRERTRTYGRIKGPVHRRGKGNRINTSALGNYHHDSKGALTAWIESKYATVEQSAEGLARSPCTGRGSPPLPTGFSIPQISGGAFHRPTNMSTSPLSDVTIESPKFSNSENAQIITAPVLQKPMGDPRSVCLPNSTVSDKQQVLQSNSIDGQISSGLQTSAIVAHDEKSGVISMNHQAPDMSCVGLKSNFQGSLHPGAVRSSWNQSLPHCFGHSNKLVEECRSSTGACTERSEILQEQHSSLQFKCSTAYNTGRYQHENLCGPAFSQQDTANEVANFSTLAFSGLVKHRQERLCKDSGSALKLGLSWVTSDSTLDLSVAKMSASQPEQSAP VAFIDFLGVGAA*Deduced amino acid sequence of CABF-3 from Physcomitrella patens (SEQ IDNO:23) MADSYGHNAGSPESSPHSDNESGGHYRDQDASVREQDRFLPIANVSRIMKKALPSNAKISKDAKETVQECVSEFISFITGEASDKCQREKRKTINGDDLLWAMSTLGFEDYVEPLKVYLHKYRELEGEKASTAKGGDQQGGKEGSQGVMGSMGMSGGMNGMNGTMNGNMHGHGIPVSMQMLQQSYGQQAPPGMMYSPHQMMPQYQMPMQSGGNQPRGV Deduced amino acidsequence of SFL-1 from Physcomitrella patens (SEQ ID NO:24)MMEAEQSYVQKLESLLGGVSTLVREEEETASVSEDEDDSNSLPQIQVAVKSKRKGERRKRRERALERAEKVATDLASAPPLPKPKKPQLAADPSDPVRAYLRDIGRTKLLTAREEVDLSHQIQDLLKLENIKSNLEREIGRNATIGEWSRAVGMEQNAFEARLKKGRFAKDKMVNSNLRLVVSIAKNYQGRGMTLQDLIQEGSMGLVRGAEKFDPTKGFKFSTYAHWWIRQAVTRSIADQSRTFRLPIHLYEVISRINKAKRMLVQEHGREARNEEVAELVGLTVEKLKSVVKSAKAPGSMERPIGKDGDTTLGELVADTDVDSPEDAIVKQLMRQDIEGVLRTLNPREREVLRLRFGLDDGRSKTLEEIGQIFKATRERIRQIEAKAMRKLRQPSRNSLLREYLDVKSDAI*

[0266]

1 79 1 986 DNA Physcomitrella patens 1 tcaagccact catccgagca tagaacatcacaacccacct tgatgatcat tctctcagcc 60 gaccagcgtc aattacgctg cgtatcgctctagcttgagg aaggcaccct cgccctcttc 120 gccgcggaag tagccctctg cttcacgagggcggcaaaac tctcccaagg cagttccggg 180 gggatgggat atagctgcag ctgctgtggggaatcctcaa aattgtacgg gatcttcttc 240 tttgtgtaga agatgccaac atcgtaggcccgggcagctt cttccggagt ttcatatgtt 300 cccagccata tcttacgttt ctgagatgtgggtcgaattt ctgtcaccca tttgtttagc 360 tcgggccggt gccgaacccc cctaaaactggtcgtatcgc cagtgttgct agcagaaact 420 tcttcggtat cccatgccga tggggccttatttaaatcaa tattccgaaa tttaaaggca 480 ttccgaccgc tagtgtcttt cgccgctaaccgcattccct tggattcttc ttccaaacta 540 gattcagact tgctctcctg ccaacttcttttttcacttt cggggattct attttagtcg 600 ttaactgcaa cgcctgttct ttgaccttgccaccacaagg atcccacttc tttgttttgg 660 gcttcccctg ttcaataatg ctggaaattgtcaaattcat gaactaccca attgcaaccc 720 ctcccaccgg gatggattga tcgccaaaatttcgtagtaa cttaactttc atacaacaac 780 ttgagttcct tcgctattag ggacacgtggcagaaacttg gacgtgcaag cgtatgtact 840 catcagagtt tgacagcgca taaaatcatataaaaagtct tgaagaagcg ttgtttaatt 900 catgggtaac cacgagttac gcggagcgtcggcagcaagg agaggacgac caggcggcaa 960 gaagatgcgt cggcaagagc tcgtgc 986 21690 DNA Physcomitrella patens 2 ttttttttgg cgaaaatggg taaaaatttccgtggccgta aaatcagttg tggcgcttgc 60 cttgcaataa gctggttatc gtaaaatggcaattaccttg atatgttcac taggttcgtg 120 tcagtgagat gccttgcaga aggcgatttcccgtttattt acaactctac atgttactga 180 agcactgtgg cattcaatct cctaacctagagatgcttaa ctgcttcgtg catgatctat 240 attacatctt gagcctcaga ctgtggcgtcttattgcaca tctagcgcta tatattacac 300 tacggtacac catcggaaga agtgaagaaggaatagctac tattttgcat ctccagtgtc 360 aaatgtgatg ctgacctgac ctaatcggcagagacgcaga tctccaatgc tcacacttca 420 catcctaaaa cgcacttgca gtgtaccctgctctcaattc ccattgcaat tccacatctg 480 ggatctaagg gcatgctttg tgggcactaaagcgccgatt tcctcctcaa tgcgaaggtt 540 gaccttactg aggaaagtgc gcatcgtaatcggcgatgta ggcttaggga tttcgccggg 600 taacactcaa cgcaatcgtg acgttcgctagatagcttgg ttgatatcag gtgcaaatcc 660 cacaaaatcc tttgcatggt cgggcatttatgtctaccag ggaagtatca gttttcttcc 720 agaataaaat ttccacttta acagcacctgctcacgaaat cctcaggcat gtggtggtgg 780 tggacggggt gaagaagaag gcccgccctcgtcaacacca tcttctccag tttggggcga 840 caccacactc ttccctcggc tcaacaaacgtcggaagctc gctgaagcac gcgccatcgg 900 cgacaaaaca ttcgacgaat tgctgacagccgctggacca gacctcggaa tgtcgatagt 960 aacctgaaac ggcgcacgaa tactggacgccgccctcgct tctgcactcc ctcctgcacc 1020 tgcactgctc atctgcgcat gattcccccagaataacaca ttggacggga ttccggacgg 1080 tgtctcgtaa tcttttatgt tttgctgctgatcaaccgca acggccgttt ccgacgtacc 1140 acttcggcga tgattctccc cgccctgatcctccgcagtg cggggcaaac tattgcctct 1200 tggggaacta ttcaacgccg gcaactgtccccggctccgt tggctcctcc tggaagctcg 1260 catggccgcc atgaacggcg ctcctacgtcacccataacg ggcgcttcca tctgaggagg 1320 ctcgcttatc tgcatcacgg tggcggcctcggtcttcttc tcagtttcat cagctcccac 1380 actcgtgcga cacaacgggc atgtcgagtgcgagtgcaac cacatgtcaa tacaatccaa 1440 gtggaaacta tggtcacact tcggcaacgtgcggcctttc tcacccaact caaattcttc 1500 caaacaaacc gcgcactcga acccacgctttgccctctca ccgtcgaatt cgaaagtggg 1560 cagagcttca ataacagccc gctcaagccccactgcctgc gtcaccggag tagcgttcac 1620 agggacagtg taacggcgtc ttcgccatgataacgtacgc aaggtgccat cgcttgcgac 1680 gatctggtgc 1690 3 502 DNAPhyscomitrella patens 3 gcaccagcgc ttttaaacaa tcaaatatct gagcaggttgatggcgaaga ttcagatgta 60 tcaagaagtg gcgctagtga tcaatctggg catgaaagaccccttgacgt tttacgcaag 120 gtgaaaggaa atgatgcttg tgccgactgc ggtgctgctgatcccgattg ggcttcgctg 180 aatcttggga ttcttctgtg tattgagtgc tcaggagtacacagaaacat gagcgttcag 240 atttctaagg tccgttcgtt gacgttagat gtcaaagtttgggagccttc tgtaatgagc 300 tattttcaat ctgtcggaaa ctcctacgct aattctatatgggaagagct tttgaatccc 360 aagtcctcag aggagtcaag tgagagaaac gttaatgacgagggacaatc gggcgtttta 420 agtgctagca gagcaaggcc aagacctaga gaccccatacctatcaaaga aagatttatc 480 aatgcaaagt atgtggagaa aa 502 4 1531 DNAPhyscomitrella patens 4 gcacgagctg ccccattcga gcccactcgc acgagaagatacgagccgcg ctttggccga 60 gtggtcgtaa gtagaagtaa aggtccgcgg ccgctgcggtctgtgaaatt ctctcgcacg 120 gagagaagcg tgttctgctg gtttcttcgc acagagacttctcctgcacc ttttcttctt 180 cctctacatc gtctcctgcg acgactacat tgtgtgggagcagtggcaac cttcctggcc 240 accccgggct tcctctcagt cagtggctca cgtctcccagctaggcctcc catcgcgtct 300 tgccggctca atcgggtgtc ttgctctgtt ttttaacctctctcccttcc ggccctctta 360 ttcctctcca gtcacttccg ccggatcgcg acttttgtacccatttgggg gttgggtgtt 420 ataagtttgc cctcagggtg tgagttgctt tgtgtgtcttttgtagtagt actttgcttg 480 ttgggtgcgg aagggaacct ttgagaagtc gacccattctctagttttgc accagtcccg 540 cttagtgtgt gtgtcattag tgttggttgc aagtctgaagccttgagcga gatttgcagg 600 attttctcat acgcttctga ttaggaaaga tacatccttattagtctgtt aaagatggcc 660 accgagcgtg tgtctcagga gacgacctcg caggcccctgagggtccagt tatgtgcaag 720 aacctttgcg gcttcttcgg cagccaagct accatggggttgtgctcgaa gtgctaccga 780 gagacagtca tgcagcgaag atgacggctt tagctgagcaagccactcag gctgctcagg 840 cgacatctgc cacagctgct gctgttcagc cccccgctcctgtacatgag accaagctca 900 catgcgaggt tgagagaaca atgattgtgc cgcatcaatcttccagctat caacaagacc 960 tggttacccc cgctgcagct gcccctcagg cagtgaagtcctctatcgca gctccctcta 1020 gacccgagcc caatcgatgc ggatcttgca ggaagcgtgttggattgaca ggatttaagt 1080 gtcgctgtgg caacctctac tgcgctttac atcggtactcggacaaacac acttgcacat 1140 atgactacaa agccgcaggg caggaagcga ttgcgaaagctaatcctctt gtcgtggccg 1200 agaaggttgt caagttttga tgagcatccg ttaagcttttctgccgacga tttaggcttc 1260 atacattgag taactctaca tctttcttct ttatcgagagagcgagtcgc atcaagatga 1320 agtcgagggg tgcgcgtcgg ttttggggag aggggatttctttccccttt ccccccttgg 1380 cggcatcgtg ttttatgtgt acagaagtag gttaggacaagatagaatca tatgccagat 1440 caattgatag tcctctttaa ggaggacact tattacacaataaaaaatcc tgggtaatgc 1500 atgccttgat tgtgttgttt tttcctcgtg c 1531 5 689DNA Physcomitrella patens 5 gcgtggggcg tctacactag tttatccccg ggctgaggaattcggcacca gatttgtcaa 60 tcaaaagaag ttagttgcgg gtgatgctat tgtatttcttcgcatcgcat ctggcgaact 120 ttgtgtcggc gtgcgccgtt caatgagggg tgtcagcaacggagaatcct catcttggca 180 ctcctcaatc agtaatgctt caacgattcg gccatctcgatgggaggtga agggcacaga 240 aagtttctcg gactttttag gtggcgttgg tgataatgggtacgcactga atagctcaat 300 tcggtctgaa aaccagggct ctccaacaac gagtagctttgcacgggacc gtgctcgtgt 360 tactgcgaag tccgttctag aagctgctgc actcgccgtctccggtgaac gttttgaggt 420 tgtgtattat cctcgtgcta gcacagctga gttctgtgtcaaagctgggc ttgttaaacg 480 tgcgctagag caatcgtggt acgctggaat gcgcttcaaaatggcatttg aaactgaaga 540 ctcctcgagg ataagctggt ttatgggaac tattgctgctgttcaagcag cagatccagt 600 aacttgtggc ctagttctcc atggcgggtc tgcaggtcaccttgggatga gcggacctat 660 tgcaggagtg atcgtgtagc catggagta 689 6 506 DNAPhyscomitrella patens 6 gcaccagtgt tccctttcat atgctcagca tgtccgccaatgagcgcgcc tgttgtgtac 60 agtctgtgga gagctgtaga aaattcaatt ccgatttcaaaatatccagc gacgatgaca 120 cggaacatgg gagtttggag gacgacatga aggagttgaacgaagacatg gaaattccct 180 taggtcgaga tggcgagggt atgcagtcaa agcagtgcccgcgcggccac tggcgtccag 240 cggaagacga caagctgcga gaactagtgt cccagtttggacctcaaaac tggaatctca 300 tagcagagaa acttcagggt cgatcaggga aaagctgcaggctacggtgg ttcaatcagc 360 tggaccctcg catcaaccgg cacccattct cggaagaagaggaagagcgg ctgcttatag 420 cacacaagcg ctacggcaac aagtgggcat tgatcgcgcgcctctttccg ggccgcacag 480 acaacgcggt gaagaatcac tggccc 506 7 536 DNAPhyscomitrella patens 7 gcaccaggtc ttcgactttg cttcagcacg cgcgcgttgtggtcgatctc tcgctggagc 60 aacaggttgt cttgtcgctg ccattgctaa agccattcttacttctagca cttctcggag 120 gttattgatt tctcgcaaat tgctcttcca cctgccctctttcgtgaggg agttcgaagc 180 tgaaaagtaa tgagctgaag attaaggtct tttacgagtgaacagcgagc acacagctag 240 caactctttc ggagaatact ccaggcgaaa ttggtcggatggccgatagc tacggtcaca 300 acgcaggttc accagagagc agcccgcatt ctgataacgagtccgggggt cattaccgag 360 accaggatgc ttctgtacgg gaacaggatc ggttcctgcccatcgcgaac gtgagccgaa 420 tcatgaagaa ggcgttgccg tctaatgcaa aaatttcgaaggacgcgaaa gagactgtgc 480 aggagtgtgt gtccgagttc atcagcttca tcactggtgaggcgtcagat aagtgc 536 8 599 DNA Physcomitrella patens modified_base(463) a, t, c, g, other or unknown 8 gcacgagttt tcttgtgtca aagcagcagaagaaatccac ttctggtagt attcaaacat 60 aaaagaatgg aaacttatgt aacagtctactttctgatcg aaacattacc aaatgccttt 120 ttcctggttt ggtaggtact atcaatcagcagcaattaaa tagcgtcaga tttcacatct 180 aagtactctc gtagaatgct gttccggctgggttgcctca gcttgcgcat cgcttttgcc 240 tcaatttgtc ttatcctttc ccgagtaactttaaagattt gacctatttc ttctaaagtc 300 ttggaccgcc catcgtccaa tccaaaacgcagtcttagca cctccctctc tcttgggttc 360 aatgtgcgta gaacgccctc tatatcttgtcgcatcaatt gctttacgat tgcgtcctca 420 ggtgaatcca catctgtgtc tgcgacaagttccccaagtg tantgtcccc atctttgcca 480 atgggccgct ccatcgaacc tggtgcctttgctgatttca ctacagattt cagtttctca 540 acagtcaagc ccactagctc agccacttcctcgttacgtg cttcccgccc atgctcctg 599 9 1057 DNA Physcomitrella patens 9gcgatatcgg aagaagaacc aagggaatgc ggttagcggc gaaagacact agcggtcgga 60atgcctttaa atttcggaat attgatttaa ataaggcccc atcggcatgg gataccgaag 120aagtttctgc tagcaacact ggcgatacga ccagttttag gggggttcgg caccggcccg 180agctaaacaa atgggtgaca gaaattcgac ccacatctca gaaacgtaag atatggctgg 240gaacatatga aactccggaa gaagctgccc gggcctacga tgttggcatc ttctacacaa 300agaagaagat cccgtacaat tttgaggatt ccccacagca gctgcagcta tatcccatcc 360ccccggaact gccttgggag agttttgccg ccctcgtgaa gcagagggct acttccgcgg 420cgaagagggc gagggtgcct tcctcaagct agagcgatac gcagcgtaat tgacgctggt 480cggctgagag aatgatcatc aaggtgggtt gtgatgttct atgctcggat gagtggcttg 540aagggttctg gttccaacca tgagagcatg acgcgagtcc cacacggatg gagcttgtga 600atggagtggt agactgtaga tggtttttgt aacggcttga gtaataacgg aagcttcatg 660gcttgaatga ccagccatgg tggtgtgcaa gtgaagatcg ctgcttgtgt gaaggtttcc 720atctttccca tccccgtctt ccactttgct acacgttgct agtgtcactt gaacaattca 780ttcatggacc ctgctctcct ttcccctgtt acgaagttct tatggtagag ttcaccgaac 840gcaagctgtc taggaagttg acagtttgtg ggagccaaaa actctacttg agctactgtg 900tgcacgcctt ctgagtcctc cagcgaggag cctgtatatt attggatggt gcaggatggg 960tcgcttggtg cctttctctt tttccttttc ctctttttgt aaatggtttt ccttctatga 1020atatgtgaag ctcctcccac ggaagcatag agctcgc 1057 10 1839 DNA Physcomitrellapatens 10 atcccgggat caggaagctg tcaaggaaga gatggaaatc ttgctccatacaattactac 60 gggccgccac cgggcagtaa caattatgtc gtcaacagca agattatggtcgtggctgtc 120 gcggttctct tcgctgtcgt cctcttcatc ctctgcctcc acatctacgccaagtggttc 180 tggcgcaatc aaggtgccat cgtcgcaagc gatggcacct tgcgtacgttatcatggcga 240 agacgccgtt acactgtccc tgtgaacgct actccggtga cgcaggcagtggggcttgag 300 cgggctgtta ttgaagctct gcccactttc gaattcgacg gtgagagggcaaagcgtgtg 360 ttcgagtgcg cggtttgttt ggaagaattt gagttgggtg agaaaggccgcacgttgccg 420 aagtgtgacc atagtttcca cttggattgt attgacatgt ggttgcactcgcactcgaca 480 tgcccgttgt gtcgcacgag tgtgggagct gatgaaactg agaagaagaccgaggccgcc 540 accgtgatgc agataagcga gcctcctcag atggaagcgc ccgttatgggtgacgtagga 600 gcgccgttca tggcggccat gcgagcttcc aggaggagcc aacggagccggggacagttg 660 ccggcgttga atagttcccc aagaggcaat agtttgcccc gcactgcggaggatcagggc 720 ggggagaatc atcgccgaag tggtacgtcg gaaacggccg ttgcggttgatcagcagcaa 780 aacataaaag attacgagac accgtccgga atcccgtcca atgtgttattctgggggaat 840 catgcgcaga tgagcagtgc aggtgcagga gggagtgcag aagcgagggcggcgtccagt 900 attcgtgcgc cgtttcaggt tactatcgac attccgaggt ctggtccagcggctgtcagc 960 aattcgtcga atgttttgtc gccgatggcg cgtgcttcag cgagcttccgacgtttgttg 1020 agccgaggga agagtgtggt gtcgccccaa actggagaag atggtgttgacgagggcggg 1080 ccttcttctt caccccgtcc accaccacca catgcctgag gatttcgtgagcaggtgctg 1140 ttaaagtgga aattttattc tggaagaaaa ctgatacttc cctggtagacataaatgccc 1200 gaccatgcaa aggattttgt gggatttgca cctgatatca accaagctatctagcgaacg 1260 tcacgattgc gttgagtgtt acccggcgaa atccctaagc ctacatcgccgattacgatg 1320 cgcactttcc tcagtaaggt caaccttcgc attgaggagg aaatcggcgctttagtgccc 1380 acaaagcatg cccttagatc ccagatgtgg aattgcaatg ggaattgagagcagggtaca 1440 ctgcaagtgc gttttaggat gtgaagtgtg agcattggag atctgcgtctctgccgatta 1500 ggtcaggtca gcatcacatt tgacactgga gatgcaaaat agtagctattccttcttcac 1560 ttcttccgat ggtgtaccgt agtgtaatat atagcgctag atgtgcaataagacgccaca 1620 gtctgaggct caagatgtaa tatagatcat gcacgaagca gttaagcatctctaggttag 1680 gagattgaat gccacagtgc ttcagtaaca tgtagagttg taaataaacgggaaatcgcc 1740 ttctgcaagg catctcactg acacgaacct agtgaacata tcaaggtaattgccatttta 1800 cgataaccag cttattgcaa ggcaagcgcc agagctcgc 1839 11 2041DNA Physcomitrella patens 11 atcccgggag gaggacttgc ggaatgcaaa atcacaatttgagcaggctc gattcaattt 60 gatgacagca cttaccaata gtgaggcaaa aaagaagttcgagttccttg aagccgtgag 120 tggtacaatg gatgcacatc tcaggtactt caagcagggctatgagttgc tacatcaaat 180 ggaaccttac atccatcagg tgttaacata tgctcaacagtccagagaaa gggccaacta 240 cgagcaagca gcacttgcag atcgtatgca ggagtacaggcaggaagttg agagagagag 300 ccaaaggtcg attgattttg acagctcttc tggagatggtattcaaggtg ttggccgcag 360 ttcacataag atgattgagg cagtcatgca atcgaccccaaaagggcaga tccagactct 420 taagcaggga tacctgttaa agcgttcaac aaatttgcgaggtgactgga agcggaggtt 480 ttttgtgttg gatagcagag gaatgctgta ttattatcggaaacagtggg gcaagcctac 540 agacgagaaa aatgtagcac atcacactgt gaatctgctgacgtctacaa tcaagataga 600 cgcagaacaa tcagatcttc gtttctgctt tcggattatttctccagcta aaagttatac 660 cctccaggca gaaaatgcca ttgacagaat ggattggatggacaaaatta caggggtgat 720 ttcgtcgctt ttaaacaatc aaatatctga gcaggttgatggcgaagatt cagatgtatc 780 aagaagtggc gctagtgatc aatctgggca tgaaagaccccttgacgttt tacgcaaggt 840 gaaaggaaat gatgcttgtg ccgactgcgg tgctgctgatcccgattggg cttcgctgaa 900 tcttgggatt cttctgtgta ttgagtgctc aggagtacacagaaacatga gcgttcagat 960 ttctaaggtc cgttcgttga cgttagatgt caaagtttgggagccttctg taatgagcta 1020 ttttcaatct gtcggaaact cctacgctaa ttctatatgggaagagcttt tgaatcccaa 1080 gtcctcagag gagtcaagtg agagaaacgt taatgacgagggacaatcgg gcgttttaag 1140 tgctagcaga gcaaggccaa gacctagaga ccccatacctatcaaagaaa gatttatcaa 1200 tgcaaagtat gtggagaaaa aatttgtcca aaagttgaaggtggattctc gaggcccgtc 1260 agtgacacgg cagatctggg atgctgtcca gaacaaaaaagtgcagcttg ctcttcgtct 1320 tcttatcact gctgatgcta acgccaacac aaccttcgagcaagtaatgg gtggtaccga 1380 gtcttcgtgg tcgtctccac ttgcaagcct cgctggagctctcttacgaa agaactctct 1440 cagtgcctct cagagtggtc gcaggaactg gagcgtaccttcactattgt cttctccaga 1500 cgatccgggg tcccgttcag gagctttaag ccctgtttcgagaagtcctg atgcagcagg 1560 cagcggaggg attgatgaga aagatttgcg gggctgcagtttgctccatg ttgcctgcca 1620 aatcggagat attagcctga tcgagctgct acttcaatacggggcgcaaa tcaattgtgt 1680 ggataccctg ggtcgaactc ctcttcatca ctgtgtcttgtgcggcaaca attcgtgtgc 1740 aaagctcctg ctcacaagag gggcgaaggc gggtgccgtagacaaagagg gaaaaactcc 1800 gctggagtgt gcagtggaga agctaggtgc tatcacggatgaagaattgt tcataatgct 1860 ttctgaaacc agtagatgac accacatttg tgcctgagttgctttgtgta taaatctcaa 1920 catcaacttg tttcctagca cctgtaaggc tagtttgtttgggtagtttg cattcttgtt 1980 ctaccgtttt atcttcccat tacgtcagca taagtagagagtggaagcag gtggatatcg 2040 c 2041 12 804 DNA Physcomitrella patens 12atcccgggca ccagtcccgc ttagtgtgtg tgtcattagt gttggttgca agtctgaagc 60cttgagcgag atttgcagga ttttctcata cgcttctgat taggaaagat acacccttat 120tagtctgtta aagatggcca ccgagcgtgt gtctcaggag acgacctcgc aggcccctga 180gggtccagtt atgtgcaaga acctttgcgg cttcttcggc agccaagcta ccatggggtt 240gtgctcgaag tgctaccgag agacagtcat gcaagcgaag atgacggctt tagctgagca 300agccactcag gctgctcagg cgacatctgc cacagctgct gctgttcagc cccccgctcc 360tgtacatgag accaagctca catgcgaggt tgagagaaca atgattgtgc cgcatcaatc 420ttccagctat caacaagacc tggttacccc cgctgcagct gcccctcagg cagtgaagtc 480ctctatcgca gctccctcta gacccgagcc caatcgatgc ggatcttgca ggaagcgtgt 540tggattgaca ggatttaagt gtcgctgtgg caacctctac tgcgctttac atcggtactc 600ggacaaacac acttgcacat atgactacaa agccgcaggg caggaagcga ttgcgaaagc 660taatcctctt gtcgtggccg agaaggttgt caagttttga tgagcatccg ttaagctttt 720ctgccgacga tttaggcttc atacattgag taactctaca tctttcttct ttatcgagag 780agcgagtcgc atcaagagct cgcc 804 13 2699 DNA Physcomitrella patens 13atcccgggta tcgatctgga gcccgttgca aactcaatgg tgtattttat agggcaaaag 60tctgatctat atggaatgca tcctctcaga gttgcaaatc atggactgca tgtcactctg 120ggttattctc gatcacctag ctttgctgga gttcaaattg gtgagtacga gtattatgag 180tgatctcgag tttatggtcc ccttctttca tgatcaaggg taatttatat caagggtgta 240tatgagagat acgcacttat tgagtggacc ttttctcata ctgcatttac acccctgtca 300gttgcagcat cctggtttgg aatgccgggt ccagtccctc tattatccat gagtgtaaaa 360tcggagagtc tcgatgacat tggaggtcac gagaaaaaat ctgtaactgg gtcggaagtg 420ggtggcctcg atgctcagct gtggcatgcc tgtgctgggg gtatggttca actgcctcat 480gtgggtgcta aggttgtcta ttttccccaa ggccatggcg aacaagctgc ttcaactccc 540gagttccccc gcactttggt tccaaatgga agtgttccct gccgagttgt gtcagttaac 600tttctggctg atacagaaac agacgaggta tttgctcgta tttgcctgca gcctgagatt 660ggctcctccg ctcaggattt aacagatgat tctcttgcgt ctccgcctct agagaaacca 720gcttcatttg ccaaaacgct cactcaaagt gatgcaaaca acggtggagg cttttcaata 780cctcgttatt gtgctgaaac tattttccca cctctcgatt actgtatcga tcctcctgtt 840caaactgttc ttgcaaaaga tgtccatgga gaggtgtgga aatttcgtca catttacagg 900gggactccac gtcgacattt gttaaccaca ggatggagca catttgtcaa tcaaaagaag 960ttagttgcgg gtgatgctat tgtatttctt cgcatcgcat ctggcgaact ttgtgtcggc 1020gtgcgccgtt caatgagggg tgtcagcaac ggagaatcct catcttggca ctcctcaatc 1080agtaatgctt caacgattcg gccatctcga tgggaggtga agggcacaga aagtttctcg 1140gactttttag gtggcgttgg tgataatggg tacgcactga atagctcaat tcggtctgaa 1200aaccagggct ctccaacaac gagtagcttt gcacgggacc gtgctcgtgt tactgcgaag 1260tccgttctag aagctgctgc actcgccgtc tccggtgaac gttttgaggt tgtgtattat 1320cctcgtgcta gcacagctga gttctgtgtc aaagctgggc ttgttaaacg tgcgctagag 1380caatcgtggt acgctggaat gcgcttcaaa atggcatttg aaactgaaga ctcctcgagg 1440ataagctggt ttatgggaac tattgctgct gttcaagcag cagatccagt actttggcct 1500agttctccat ggcgggttct gcaggtcact tgggatgagc cggacctatt gcagggagtg 1560aatcgtgtaa gcccatggca gttagagctt gtggcgacac ttcctatgca gctgccccct 1620gtctctcttc ccaaaaagaa actgcgcact gtccagcctc aagagcttcc acttcagccc 1680cctggactgc taagcctgcc gttggcaggg actagcaact ttggtgggca cttggccacc 1740ccctggggca gctctgttct tttggatgac gcctctgttg gcatgcaggg ggccaggcat 1800gatcaattca acgggcttcc aactgtggat ttccgaaata gtaactacaa acatcctcgg 1860gagttttcta gggacaatca gtaccagatt caagatcatc aagtcttcca tcctagacct 1920gtattaaatg agccccctgc gacaaacact ggcaactact tctctctttt acctagtctc 1980cagcgacggc cagatatctc tcctagtatt cagcccttag ccttcatgtc tgcttctgga 2040agctcacagc tggagacttc ttcaactaag acagcggcca cctctttttt cctatttggc 2100caattcattg acccttcttg cacctccaaa cctcagcagc gttccacagt tattaataac 2160gcttccgttg ctggggatgg taagcatcct ggcactaata actcatcctc ggataacaaa 2220tcagaggaca aggacaattg tagggatgtt caacccattc tgaatgggat tgctgtaaga 2280tctggatttc gagcagatat agccgcgaag aagtttcaac agagcgactc tgcacatccc 2340acggaagcat cacgtggaag ccaagttagc agcttaccgt ggtggcaaac acaggacgct 2400cacaaggatc aggaattcca tggagacagt cagacgcctc atactcctgc atctggtagc 2460caatgaggct aaagcttgat catagctcat aaccctctca caggacgtaa tgggggtgac 2520aacatgctaa cagaattgca cggtaaagga aaactgtact aggcatgtta tatgggaatt 2580cggatcgctt cttgcaatta aacacgctag cgccgtttgg tgccaatgtt attctggcat 2640ttgttttgtt tcctttggaa acaaattgct atatttcaaa gtcctttgga ggagctcgc 2699 142367 DNA Physcomitrella patens 14 atcccgggct gttgtgtaca gtctgtggagagctgtagaa aattcaattc cgatttcaaa 60 atatccagcg acgatgacac ggaacatgggagtttggagg acgacatgaa ggagttgaac 120 gaagacatgg aaattccctt aggtcgagatggcgagggta tgcagtcaaa gcagtgcccg 180 cgcggccact ggcgtccagc ggaagacgacaagttgcgag aactagtgtc ccagtttgga 240 cctcaaaact ggaatctcat agcagagaaacttcagggtc gatcagggaa aagctgcagg 300 ctacggtggt tcaatcagct ggaccctcgcatcaaccggc acccattctc ggaagaagag 360 gaagagcggc tgcttatagc acacaagcgctacggcaaca agtgggcatt gatcgcgcgc 420 ctctttccgg gccgcacaga caacgcggtgaagaatcact ggcacgttgt gacggcaaga 480 cagtcccgtg aacggacacg aacttacggccgtatcaaag gtccggtaca tcgaagaggc 540 aagggtaacc gtatcaatac ctccgcacttggaaattacc atcacgattc gaagggagct 600 ctcacagcct ggattgagtc gaagtatgcgacagtcgagc agtctgcgga agggctcgct 660 aggtctcctt gtaccggcag aggctctcctcctctaccca ccggtttcag tataccgcag 720 atttccggcg gcgccttcca tcgaccgacaaacatgagta ctagtcctct tagcgatgtg 780 actatcgagt cgccaaagtt tagcaactccgaaaatgcgc aaataataac cgcgcccgtc 840 ctgcaaaagc caatgggaga tcccaggtcagtatgcttgc cgaattcgac tgtttccgac 900 aagcagcaag tgctgcagag taattccatcgacggtcaga tctcctccgg gctccagaca 960 agcgcaatag tagcgcatga tgagaaatcgggcgtcattt caatgaatca tcaagcaccg 1020 gatatgtcct gtgttggatt gaagtcaaattttcagggga gtctccatcc tggcgctgtt 1080 agatcttctt ggaatcaatc ccttccccactgttttggcc acagtaacaa gttggtggag 1140 gagtgcagga gttctacagg cgcatgcactgaacgctctg agattctgca agaacagcat 1200 tctagccttc agtttaaatg cagcactgcgtacaatactg gaagatatca acatgaaaac 1260 ctttgtgggc cagcattctc gcaacaagacacagcgaacg aggttgcgaa tttttctacg 1320 ttggcattct ccggcctagt gaagcatcgccaagagaggt tgtgcaaaga tagtggatct 1380 gctctcaagc tgggactatc atgggttacatccgatagca ctcttgactt gagtgttgcc 1440 aaaatgtcag catcgcagcc agagcagtctgcgccggttg cattcattga ttttctaggc 1500 gtgggagcgg cctgaaggct gcggaaagattttagcaaag cttttataac gttttttttg 1560 cacagggctg tttttagctt gtataccagtaggcacttct acttcttttt cttcttttct 1620 ttttcccctt ttcttctccc cccactttcaccatttccgc catagcagcc tttgaatcac 1680 gtaatggaac ctttggcggc ctgtatgaggcacttttgga ggcatccctg gacgaagaat 1740 ggatcaaacc gtactgcgga tgtcatgctttgaagctgca atccgaattc agtagcatgc 1800 tgtggatgac tcaaaaggag tagctgctttgtgaaactaa tactatacag cggattttga 1860 agacccaagt ttcatgtgga caagtctgaaaaacttatac gccacctcca tgggcttcta 1920 cgatgaatat gcgctttcgg cttacactgcggctcttttt tgcatatata tatacttcca 1980 ttcaatttta tttggaaatg ttttgaatctaccttctcgt acaaaactgg gatcagaaat 2040 cttccaggtt gtgggtcgca agttaactctgcagattgtg gctgacactt gggcaatcgg 2100 caactttatc tttttgtttt ttacgcttgaacggacctca gctgtacaga cactcatcat 2160 gtacattcga tgccatctct tggctttcatggaagttcag atatcggaaa ctgtgacaga 2220 gacagagaga gagagagaga gagagagagagagattcttg atgcactgtg cgccgagttt 2280 gagactagtt tagaaagatt gatgaagctagcagtaaatt gttggcctca tctgaaaggt 2340 acggccttta ctccgtgagc ccgggat 236715 787 DNA Physcomitrella patens 15 atcccgggca gcgagcacac agctagcaactctttcggag aatactccag gcgaaattgg 60 tcggatggcc gatagctacg gtcacaacgcaggttcacca gagagcagcc cgcattctga 120 taacgagtcc gggggtcatt accgagaccaggatgcttct gtacgggaac aggatcggtt 180 cctgcccatc gcgaacgtga gccgaatcatgaagaaggcg ttgccgtcta atgcaaaaat 240 ttcgaaggac gcgaaagaga ctgtgcaggagtgtgtgtcc gagttcatca gcttcatcac 300 tggtgaggcg tcagataagt gccagagggagaagagaaag acgatcaacg gtgacgactt 360 gctgtgggcc atgagtacac ttggtttcgaagattacgtg gagcctctga aggtttacct 420 acacaaatac cgggagctag agggagagaaggcttccacg gccaagggtg gtgaccagca 480 aggagggaaa gaagggagtc aaggtgttatggggtccatg ggtatgtcgg gcggaatgaa 540 cggtatgaac ggtacgatga acgggaatatgcatggacat ggaatcccgg tgtcgatgca 600 gatgctgcag cagtcgtacg gacagcaggcacctccaggg atgatgtatt cccctcatca 660 gatgatgccg caataccaga tgccaatgcagtctggtgga aaccagcctc gtggagtgta 720 ggaggttcca cggcgaggag aatttgaaattggggagatt gtcaaccgcg tgagggagtg 780 agctcgc 787 16 1669 DNAPhyscomitrella patens 16 atcccgggct cggaaggact gtgcattgtc gagcgctgaaggtggatgat gctttggtga 60 ccgagagcgg tcttatcagt gaagaaggag tttctcgtgctgcagctgag gaggcgatga 120 cgttagcttt agcagctgca aaggccgcca tggaggctgcctcgtacgct gatgcgatgc 180 cgtggaacag gaggagtttc cgacggaatt tgatctgctgagactagaga gggccaggtt 240 gagcgatgtt gagcattctt ttcgggttga attggatacagaggctgcca tgatggaggc 300 cgagcagagt tatgtgcaga agctagaatc gttgttgggaggtgtttcca cgctcgtccg 360 tgaggaagag gaaactgcat ccgtttcaga agatgaagatgattcaaaca gcttacctca 420 aattcaagta gccgttaaat cgaagcggaa gggagagaggaggaagaggc gggagcgagc 480 gttggaaagg gcagagaagg ttgccaccga tcttgcatcagcaccccctc tcccaaaacc 540 taagaaacca cagcttgcgg cggatccttc agacccagtccgtgcatatt tgcgagacat 600 aggaaggacg aagttgctaa cagcaagaga agaagtcgatctctctcatc aaattcagga 660 tcttttgaag ttggagaata tcaagtctaa ccttgagcgagagataggaa ggaatgccac 720 aattggagag tggagtagag cggtaggaat ggaacagaatgcatttgaag cgcggcttaa 780 gaagggtcga ttcgccaagg acaaaatggt gaattcgaatttgcggttgg ttgtctcgat 840 tgcgaaaaac taccagggcc gaggcatgac tcttcaagatttaattcagg aagggagcat 900 gggattggtg agaggagcgg agaagttcga cccgaccaaggggtttaagt tcagcactta 960 cgcacattgg tggattaggc aggctgtaac gcgatcaattgcggatcaat ctaggacttt 1020 tcgtttacct attcatttat acgaagttat ctcacgtatcaacaaagcaa agcgaatgct 1080 ggttcaggag catgggcggg aagcacgtaa cgaggaagtggctgagctag tgggcttgac 1140 tgttgagaaa ctgaaatctg tagtgaaatc agcaaaggcaccaggttcga tggagcggcc 1200 cattggcaaa gatggggaca ctacacttgg ggaacttgtcgcagacacag atgtggattc 1260 acctgaggac gcaatcgtaa agcaattgat gcgacaagatatagagggcg ttctacgcac 1320 attgaaccca agagagaggg aggtgctaag actgcgttttggattggacg atgggcggtc 1380 caagacttta gaagaaatag gtcaaatctt taaagctactcgggaaagga taagacaaat 1440 tgaggcaaaa gcgatgcgca agctgaggca acccagccggaacagcattc tacgagagta 1500 cttagatgtg aaatctgacg ctatttaatt gctgctgattgatagtacct accaaaccag 1560 gaaaaaggca tttggtaatg tttcgatcag aaagtagactgttacataag tttccattct 1620 tttatgtttg aatactacca gaagtggatt tcttctgctgcgagctcgc 1669 17 141 PRT Physcomitrella patens 17 Met Arg Leu Ala AlaLys Asp Thr Ser Gly Arg Asn Ala Phe Lys Phe 1 5 10 15 Arg Asn Ile AspLeu Asn Lys Ala Pro Ser Ala Trp Asp Thr Glu Glu 20 25 30 Val Ser Ala SerAsn Thr Gly Asp Thr Thr Ser Phe Arg Gly Val Arg 35 40 45 His Arg Pro GluLeu Asn Lys Trp Val Thr Glu Ile Arg Pro Thr Ser 50 55 60 Gln Lys Arg LysIle Trp Leu Gly Thr Tyr Glu Thr Pro Glu Glu Ala 65 70 75 80 Ala Arg AlaTyr Asp Val Gly Ile Phe Tyr Thr Lys Lys Lys Ile Pro 85 90 95 Tyr Asn PheGlu Asp Ser Pro Gln Gln Leu Gln Leu Tyr Pro Ile Pro 100 105 110 Pro GluLeu Pro Trp Glu Ser Phe Ala Ala Leu Val Lys Gln Arg Ala 115 120 125 ThrSer Ala Ala Lys Arg Ala Arg Val Pro Ser Ser Ser 130 135 140 18 337 PRTPhyscomitrella patens 18 Met Val Val Ala Val Ala Val Leu Phe Ala Val ValLeu Phe Ile Leu 1 5 10 15 Cys Leu His Ile Tyr Ala Lys Trp Phe Trp ArgAsn Gln Gly Ala Ile 20 25 30 Val Ala Ser Asp Gly Thr Leu Arg Thr Leu SerTrp Arg Arg Arg Arg 35 40 45 Tyr Thr Val Pro Val Asn Ala Thr Pro Val ThrGln Ala Val Gly Leu 50 55 60 Glu Arg Ala Val Ile Glu Ala Leu Pro Thr PheGlu Phe Asp Gly Glu 65 70 75 80 Arg Ala Lys Arg Val Phe Glu Cys Ala ValCys Leu Glu Glu Phe Glu 85 90 95 Leu Gly Glu Lys Gly Arg Thr Leu Pro LysCys Asp His Ser Phe His 100 105 110 Leu Asp Cys Ile Asp Met Trp Leu HisSer His Ser Thr Cys Pro Leu 115 120 125 Cys Arg Thr Ser Val Gly Ala AspGlu Thr Glu Lys Lys Thr Glu Ala 130 135 140 Ala Thr Val Met Gln Ile SerGlu Pro Pro Gln Met Glu Ala Pro Val 145 150 155 160 Met Gly Asp Val GlyAla Pro Phe Met Ala Ala Met Arg Ala Ser Arg 165 170 175 Arg Ser Gln ArgSer Arg Gly Gln Leu Pro Ala Leu Asn Ser Ser Pro 180 185 190 Arg Gly AsnSer Leu Pro Arg Thr Ala Glu Asp Gln Gly Gly Glu Asn 195 200 205 His ArgArg Ser Gly Thr Ser Glu Thr Ala Val Ala Val Asp Gln Gln 210 215 220 GlnAsn Ile Lys Asp Tyr Glu Thr Pro Ser Gly Ile Pro Ser Asn Val 225 230 235240 Leu Phe Trp Gly Asn His Ala Gln Met Ser Ser Ala Gly Ala Gly Gly 245250 255 Ser Ala Glu Ala Arg Ala Ala Ser Ser Ile Arg Ala Pro Phe Gln Val260 265 270 Thr Ile Asp Ile Pro Arg Ser Gly Pro Ala Ala Val Ser Asn SerSer 275 280 285 Asn Val Leu Ser Pro Met Ala Arg Ala Ser Ala Ser Phe ArgArg Leu 290 295 300 Leu Ser Arg Gly Lys Ser Val Val Ser Pro Gln Thr GlyGlu Asp Gly 305 310 315 320 Val Asp Glu Gly Gly Pro Ser Ser Ser Pro ArgPro Pro Pro Pro His 325 330 335 Ala 19 605 PRT Physcomitrella patens 19Met Thr Ala Leu Thr Asn Ser Glu Ala Lys Lys Lys Phe Glu Phe Leu 1 5 1015 Glu Ala Val Ser Gly Thr Met Asp Ala His Leu Arg Tyr Phe Lys Gln 20 2530 Gly Tyr Glu Leu Leu His Gln Met Glu Pro Tyr Ile His Gln Val Leu 35 4045 Thr Tyr Ala Gln Gln Ser Arg Glu Arg Ala Asn Tyr Glu Gln Ala Ala 50 5560 Leu Ala Asp Arg Met Gln Glu Tyr Arg Gln Glu Val Glu Arg Glu Ser 65 7075 80 Gln Arg Ser Ile Asp Phe Asp Ser Ser Ser Gly Asp Gly Ile Gln Gly 8590 95 Val Gly Arg Ser Ser His Lys Met Ile Glu Ala Val Met Gln Ser Thr100 105 110 Pro Lys Gly Gln Ile Gln Thr Leu Lys Gln Gly Tyr Leu Leu LysArg 115 120 125 Ser Thr Asn Leu Arg Gly Asp Trp Lys Arg Arg Phe Phe ValLeu Asp 130 135 140 Ser Arg Gly Met Leu Tyr Tyr Tyr Arg Lys Gln Trp GlyLys Pro Thr 145 150 155 160 Asp Glu Lys Asn Val Ala His His Thr Val AsnLeu Leu Thr Ser Thr 165 170 175 Ile Lys Ile Asp Ala Glu Gln Ser Asp LeuArg Phe Cys Phe Arg Ile 180 185 190 Ile Ser Pro Ala Lys Ser Tyr Thr LeuGln Ala Glu Asn Ala Ile Asp 195 200 205 Arg Met Asp Trp Met Asp Lys IleThr Gly Val Ile Ser Ser Leu Leu 210 215 220 Asn Asn Gln Ile Ser Glu GlnVal Asp Gly Glu Asp Ser Asp Val Ser 225 230 235 240 Arg Ser Gly Ala SerAsp Gln Ser Gly His Glu Arg Pro Leu Asp Val 245 250 255 Leu Arg Lys ValLys Gly Asn Asp Ala Cys Ala Asp Cys Gly Ala Ala 260 265 270 Asp Pro AspTrp Ala Ser Leu Asn Leu Gly Ile Leu Leu Cys Ile Glu 275 280 285 Cys SerGly Val His Arg Asn Met Ser Val Gln Ile Ser Lys Val Arg 290 295 300 SerLeu Thr Leu Asp Val Lys Val Trp Glu Pro Ser Val Met Ser Tyr 305 310 315320 Phe Gln Ser Val Gly Asn Ser Tyr Ala Asn Ser Ile Trp Glu Glu Leu 325330 335 Leu Asn Pro Lys Ser Ser Glu Glu Ser Ser Glu Arg Asn Val Asn Asp340 345 350 Glu Gly Gln Ser Gly Val Leu Ser Ala Ser Arg Ala Arg Pro ArgPro 355 360 365 Arg Asp Pro Ile Pro Ile Lys Glu Arg Phe Ile Asn Ala LysTyr Val 370 375 380 Glu Lys Lys Phe Val Gln Lys Leu Lys Val Asp Ser ArgGly Pro Ser 385 390 395 400 Val Thr Arg Gln Ile Trp Asp Ala Val Gln AsnLys Lys Val Gln Leu 405 410 415 Ala Leu Arg Leu Leu Ile Thr Ala Asp AlaAsn Ala Asn Thr Thr Phe 420 425 430 Glu Gln Val Met Gly Gly Thr Glu SerSer Trp Ser Ser Pro Leu Ala 435 440 445 Ser Leu Ala Gly Ala Leu Leu ArgLys Asn Ser Leu Ser Ala Ser Gln 450 455 460 Ser Gly Arg Arg Asn Trp SerVal Pro Ser Leu Leu Ser Ser Pro Asp 465 470 475 480 Asp Pro Gly Ser ArgSer Gly Ala Leu Ser Pro Val Ser Arg Ser Pro 485 490 495 Asp Ala Ala GlySer Gly Gly Ile Asp Glu Lys Asp Leu Arg Gly Cys 500 505 510 Ser Leu LeuHis Val Ala Cys Gln Ile Gly Asp Ile Ser Leu Ile Glu 515 520 525 Leu LeuLeu Gln Tyr Gly Ala Gln Ile Asn Cys Val Asp Thr Leu Gly 530 535 540 ArgThr Pro Leu His His Cys Val Leu Cys Gly Asn Asn Ser Cys Ala 545 550 555560 Lys Leu Leu Leu Thr Arg Gly Ala Lys Ala Gly Ala Val Asp Lys Glu 565570 575 Gly Lys Thr Pro Leu Glu Cys Ala Val Glu Lys Leu Gly Ala Ile Thr580 585 590 Asp Glu Glu Leu Phe Ile Met Leu Ser Glu Thr Ser Arg 595 600605 20 188 PRT Physcomitrella patens 20 Met Ala Thr Glu Arg Val Ser GlnGlu Thr Thr Ser Gln Ala Pro Glu 1 5 10 15 Gly Pro Val Met Cys Lys AsnLeu Cys Gly Phe Phe Gly Ser Gln Ala 20 25 30 Thr Met Gly Leu Cys Ser LysCys Tyr Arg Glu Thr Val Met Gln Ala 35 40 45 Lys Met Thr Ala Leu Ala GluGln Ala Thr Gln Ala Ala Gln Ala Thr 50 55 60 Ser Ala Thr Ala Ala Ala ValGln Pro Pro Ala Pro Val His Glu Thr 65 70 75 80 Lys Leu Thr Cys Glu ValGlu Arg Thr Met Ile Val Pro His Gln Ser 85 90 95 Ser Ser Tyr Gln Gln AspLeu Val Thr Pro Ala Ala Ala Ala Pro Gln 100 105 110 Ala Val Lys Ser SerIle Ala Ala Pro Ser Arg Pro Glu Pro Asn Arg 115 120 125 Cys Gly Ser CysArg Lys Arg Val Gly Leu Thr Gly Phe Lys Cys Arg 130 135 140 Cys Gly AsnLeu Tyr Cys Ala Leu His Arg Tyr Ser Asp Lys His Thr 145 150 155 160 CysThr Tyr Asp Tyr Lys Ala Ala Gly Gln Glu Ala Ile Ala Lys Ala 165 170 175Asn Pro Leu Val Val Ala Glu Lys Val Val Lys Phe 180 185 21 714 PRTPhyscomitrella patens 21 Met Pro Gly Pro Val Pro Leu Leu Ser Met Ser ValLys Ser Glu Ser 1 5 10 15 Leu Asp Asp Ile Gly Gly His Glu Lys Lys SerVal Thr Gly Ser Glu 20 25 30 Val Gly Gly Leu Asp Ala Gln Leu Trp His AlaCys Ala Gly Gly Met 35 40 45 Val Gln Leu Pro His Val Gly Ala Lys Val ValTyr Phe Pro Gln Gly 50 55 60 His Gly Glu Gln Ala Ala Ser Thr Pro Glu PhePro Arg Thr Leu Val 65 70 75 80 Pro Asn Gly Ser Val Pro Cys Arg Val ValSer Val Asn Phe Leu Ala 85 90 95 Asp Thr Glu Thr Asp Glu Val Phe Ala ArgIle Cys Leu Gln Pro Glu 100 105 110 Ile Gly Ser Ser Ala Gln Asp Leu ThrAsp Asp Ser Leu Ala Ser Pro 115 120 125 Pro Leu Glu Lys Pro Ala Ser PheAla Lys Thr Leu Thr Gln Ser Asp 130 135 140 Ala Asn Asn Gly Gly Gly PheSer Ile Pro Arg Tyr Cys Ala Glu Thr 145 150 155 160 Ile Phe Pro Pro LeuAsp Tyr Cys Ile Asp Pro Pro Val Gln Thr Val 165 170 175 Leu Ala Lys AspVal His Gly Glu Val Trp Lys Phe Arg His Ile Tyr 180 185 190 Arg Gly ThrPro Arg Arg His Leu Leu Thr Thr Gly Trp Ser Thr Phe 195 200 205 Val AsnGln Lys Lys Leu Val Ala Gly Asp Ala Ile Val Phe Leu Arg 210 215 220 IleAla Ser Gly Glu Leu Cys Val Gly Val Arg Arg Ser Met Arg Gly 225 230 235240 Val Ser Asn Gly Glu Ser Ser Ser Trp His Ser Ser Ile Ser Asn Ala 245250 255 Ser Thr Ile Arg Pro Ser Arg Trp Glu Val Lys Gly Thr Glu Ser Phe260 265 270 Ser Asp Phe Leu Gly Gly Val Gly Asp Asn Gly Tyr Ala Leu AsnSer 275 280 285 Ser Ile Arg Ser Glu Asn Gln Gly Ser Pro Thr Thr Ser SerPhe Ala 290 295 300 Arg Asp Arg Ala Arg Val Thr Ala Lys Ser Val Leu GluAla Ala Ala 305 310 315 320 Leu Ala Val Ser Gly Glu Arg Phe Glu Val ValTyr Tyr Pro Arg Ala 325 330 335 Ser Thr Ala Glu Phe Cys Val Lys Ala GlyLeu Val Lys Arg Ala Leu 340 345 350 Glu Gln Ser Trp Tyr Ala Gly Met ArgPhe Lys Met Ala Phe Glu Thr 355 360 365 Glu Asp Ser Ser Arg Ile Ser TrpPhe Met Gly Thr Ile Ala Ala Val 370 375 380 Gln Ala Ala Asp Pro Val LeuTrp Pro Ser Ser Pro Trp Arg Val Leu 385 390 395 400 Gln Val Thr Trp AspGlu Pro Asp Leu Leu Gln Gly Val Asn Arg Val 405 410 415 Ser Pro Trp GlnLeu Glu Leu Val Ala Thr Leu Pro Met Gln Leu Pro 420 425 430 Pro Val SerLeu Pro Lys Lys Lys Leu Arg Thr Val Gln Pro Gln Glu 435 440 445 Leu ProLeu Gln Pro Pro Gly Leu Leu Ser Leu Pro Leu Ala Gly Thr 450 455 460 SerAsn Phe Gly Gly His Leu Ala Thr Pro Trp Gly Ser Ser Val Leu 465 470 475480 Leu Asp Asp Ala Ser Val Gly Met Gln Gly Ala Arg His Asp Gln Phe 485490 495 Asn Gly Leu Pro Thr Val Asp Phe Arg Asn Ser Asn Tyr Lys His Pro500 505 510 Arg Glu Phe Ser Arg Asp Asn Gln Tyr Gln Ile Gln Asp His GlnVal 515 520 525 Phe His Pro Arg Pro Val Leu Asn Glu Pro Pro Ala Thr AsnThr Gly 530 535 540 Asn Tyr Phe Ser Leu Leu Pro Ser Leu Gln Arg Arg ProAsp Ile Ser 545 550 555 560 Pro Ser Ile Gln Pro Leu Ala Phe Met Ser AlaSer Gly Ser Ser Gln 565 570 575 Leu Glu Thr Ser Ser Thr Lys Thr Ala AlaThr Ser Phe Phe Leu Phe 580 585 590 Gly Gln Phe Ile Asp Pro Ser Cys ThrSer Lys Pro Gln Gln Arg Ser 595 600 605 Thr Val Ile Asn Asn Ala Ser ValAla Gly Asp Gly Lys His Pro Gly 610 615 620 Thr Asn Asn Ser Ser Ser AspAsn Lys Ser Glu Asp Lys Asp Asn Cys 625 630 635 640 Arg Asp Val Gln ProIle Leu Asn Gly Ile Ala Val Arg Ser Gly Phe 645 650 655 Arg Ala Asp IleAla Ala Lys Lys Phe Gln Gln Ser Asp Ser Ala His 660 665 670 Pro Thr GluAla Ser Arg Gly Ser Gln Val Ser Ser Leu Pro Trp Trp 675 680 685 Gln ThrGln Asp Ala His Lys Asp Gln Glu Phe His Gly Asp Ser Gln 690 695 700 ThrPro His Thr Pro Ala Ser Gly Ser Gln 705 710 22 469 PRT Physcomitrellapatens 22 Met Lys Glu Leu Asn Glu Asp Met Glu Ile Pro Leu Gly Arg AspGly 1 5 10 15 Glu Gly Met Gln Ser Lys Gln Cys Pro Arg Gly His Trp ArgPro Ala 20 25 30 Glu Asp Asp Lys Leu Arg Glu Leu Val Ser Gln Phe Gly ProGln Asn 35 40 45 Trp Asn Leu Ile Ala Glu Lys Leu Gln Gly Arg Ser Gly LysSer Cys 50 55 60 Arg Leu Arg Trp Phe Asn Gln Leu Asp Pro Arg Ile Asn ArgHis Pro 65 70 75 80 Phe Ser Glu Glu Glu Glu Glu Arg Leu Leu Ile Ala HisLys Arg Tyr 85 90 95 Gly Asn Lys Trp Ala Leu Ile Ala Arg Leu Phe Pro GlyArg Thr Asp 100 105 110 Asn Ala Val Lys Asn His Trp His Val Val Thr AlaArg Gln Ser Arg 115 120 125 Glu Arg Thr Arg Thr Tyr Gly Arg Ile Lys GlyPro Val His Arg Arg 130 135 140 Gly Lys Gly Asn Arg Ile Asn Thr Ser AlaLeu Gly Asn Tyr His His 145 150 155 160 Asp Ser Lys Gly Ala Leu Thr AlaTrp Ile Glu Ser Lys Tyr Ala Thr 165 170 175 Val Glu Gln Ser Ala Glu GlyLeu Ala Arg Ser Pro Cys Thr Gly Arg 180 185 190 Gly Ser Pro Pro Leu ProThr Gly Phe Ser Ile Pro Gln Ile Ser Gly 195 200 205 Gly Ala Phe His ArgPro Thr Asn Met Ser Thr Ser Pro Leu Ser Asp 210 215 220 Val Thr Ile GluSer Pro Lys Phe Ser Asn Ser Glu Asn Ala Gln Ile 225 230 235 240 Ile ThrAla Pro Val Leu Gln Lys Pro Met Gly Asp Pro Arg Ser Val 245 250 255 CysLeu Pro Asn Ser Thr Val Ser Asp Lys Gln Gln Val Leu Gln Ser 260 265 270Asn Ser Ile Asp Gly Gln Ile Ser Ser Gly Leu Gln Thr Ser Ala Ile 275 280285 Val Ala His Asp Glu Lys Ser Gly Val Ile Ser Met Asn His Gln Ala 290295 300 Pro Asp Met Ser Cys Val Gly Leu Lys Ser Asn Phe Gln Gly Ser Leu305 310 315 320 His Pro Gly Ala Val Arg Ser Ser Trp Asn Gln Ser Leu ProHis Cys 325 330 335 Phe Gly His Ser Asn Lys Leu Val Glu Glu Cys Arg SerSer Thr Gly 340 345 350 Ala Cys Thr Glu Arg Ser Glu Ile Leu Gln Glu GlnHis Ser Ser Leu 355 360 365 Gln Phe Lys Cys Ser Thr Ala Tyr Asn Thr GlyArg Tyr Gln His Glu 370 375 380 Asn Leu Cys Gly Pro Ala Phe Ser Gln GlnAsp Thr Ala Asn Glu Val 385 390 395 400 Ala Asn Phe Ser Thr Leu Ala PheSer Gly Leu Val Lys His Arg Gln 405 410 415 Glu Arg Leu Cys Lys Asp SerGly Ser Ala Leu Lys Leu Gly Leu Ser 420 425 430 Trp Val Thr Ser Asp SerThr Leu Asp Leu Ser Val Ala Lys Met Ser 435 440 445 Ala Ser Gln Pro GluGln Ser Ala Pro Val Ala Phe Ile Asp Phe Leu 450 455 460 Gly Val Gly AlaAla 465 23 218 PRT Physcomitrella patens 23 Met Ala Asp Ser Tyr Gly HisAsn Ala Gly Ser Pro Glu Ser Ser Pro 1 5 10 15 His Ser Asp Asn Glu SerGly Gly His Tyr Arg Asp Gln Asp Ala Ser 20 25 30 Val Arg Glu Gln Asp ArgPhe Leu Pro Ile Ala Asn Val Ser Arg Ile 35 40 45 Met Lys Lys Ala Leu ProSer Asn Ala Lys Ile Ser Lys Asp Ala Lys 50 55 60 Glu Thr Val Gln Glu CysVal Ser Glu Phe Ile Ser Phe Ile Thr Gly 65 70 75 80 Glu Ala Ser Asp LysCys Gln Arg Glu Lys Arg Lys Thr Ile Asn Gly 85 90 95 Asp Asp Leu Leu TrpAla Met Ser Thr Leu Gly Phe Glu Asp Tyr Val 100 105 110 Glu Pro Leu LysVal Tyr Leu His Lys Tyr Arg Glu Leu Glu Gly Glu 115 120 125 Lys Ala SerThr Ala Lys Gly Gly Asp Gln Gln Gly Gly Lys Glu Gly 130 135 140 Ser GlnGly Val Met Gly Ser Met Gly Met Ser Gly Gly Met Asn Gly 145 150 155 160Met Asn Gly Thr Met Asn Gly Asn Met His Gly His Gly Ile Pro Val 165 170175 Ser Met Gln Met Leu Gln Gln Ser Tyr Gly Gln Gln Ala Pro Pro Gly 180185 190 Met Met Tyr Ser Pro His Gln Met Met Pro Gln Tyr Gln Met Pro Met195 200 205 Gln Ser Gly Gly Asn Gln Pro Arg Gly Val 210 215 24 412 PRTPhyscomitrella patens 24 Met Met Glu Ala Glu Gln Ser Tyr Val Gln Lys LeuGlu Ser Leu Leu 1 5 10 15 Gly Gly Val Ser Thr Leu Val Arg Glu Glu GluGlu Thr Ala Ser Val 20 25 30 Ser Glu Asp Glu Asp Asp Ser Asn Ser Leu ProGln Ile Gln Val Ala 35 40 45 Val Lys Ser Lys Arg Lys Gly Glu Arg Arg LysArg Arg Glu Arg Ala 50 55 60 Leu Glu Arg Ala Glu Lys Val Ala Thr Asp LeuAla Ser Ala Pro Pro 65 70 75 80 Leu Pro Lys Pro Lys Lys Pro Gln Leu AlaAla Asp Pro Ser Asp Pro 85 90 95 Val Arg Ala Tyr Leu Arg Asp Ile Gly ArgThr Lys Leu Leu Thr Ala 100 105 110 Arg Glu Glu Val Asp Leu Ser His GlnIle Gln Asp Leu Leu Lys Leu 115 120 125 Glu Asn Ile Lys Ser Asn Leu GluArg Glu Ile Gly Arg Asn Ala Thr 130 135 140 Ile Gly Glu Trp Ser Arg AlaVal Gly Met Glu Gln Asn Ala Phe Glu 145 150 155 160 Ala Arg Leu Lys LysGly Arg Phe Ala Lys Asp Lys Met Val Asn Ser 165 170 175 Asn Leu Arg LeuVal Val Ser Ile Ala Lys Asn Tyr Gln Gly Arg Gly 180 185 190 Met Thr LeuGln Asp Leu Ile Gln Glu Gly Ser Met Gly Leu Val Arg 195 200 205 Gly AlaGlu Lys Phe Asp Pro Thr Lys Gly Phe Lys Phe Ser Thr Tyr 210 215 220 AlaHis Trp Trp Ile Arg Gln Ala Val Thr Arg Ser Ile Ala Asp Gln 225 230 235240 Ser Arg Thr Phe Arg Leu Pro Ile His Leu Tyr Glu Val Ile Ser Arg 245250 255 Ile Asn Lys Ala Lys Arg Met Leu Val Gln Glu His Gly Arg Glu Ala260 265 270 Arg Asn Glu Glu Val Ala Glu Leu Val Gly Leu Thr Val Glu LysLeu 275 280 285 Lys Ser Val Val Lys Ser Ala Lys Ala Pro Gly Ser Met GluArg Pro 290 295 300 Ile Gly Lys Asp Gly Asp Thr Thr Leu Gly Glu Leu ValAla Asp Thr 305 310 315 320 Asp Val Asp Ser Pro Glu Asp Ala Ile Val LysGln Leu Met Arg Gln 325 330 335 Asp Ile Glu Gly Val Leu Arg Thr Leu AsnPro Arg Glu Arg Glu Val 340 345 350 Leu Arg Leu Arg Phe Gly Leu Asp AspGly Arg Ser Lys Thr Leu Glu 355 360 365 Glu Ile Gly Gln Ile Phe Lys AlaThr Arg Glu Arg Ile Arg Gln Ile 370 375 380 Glu Ala Lys Ala Met Arg LysLeu Arg Gln Pro Ser Arg Asn Ser Ile 385 390 395 400 Leu Arg Glu Tyr LeuAsp Val Lys Ser Asp Ala Ile 405 410 25 18 DNA Artificial SequenceDescription of Artificial Sequence Primer 25 caggaaacag ctatgacc 18 2619 DNA Artificial Sequence Description of Artificial Sequence Primer 26ctaaagggaa caaaagctg 19 27 18 DNA Artificial Sequence Description ofArtificial Sequence Primer 27 tgtaaaacga cggccagt 18 28 34 DNAArtificial Sequence Description of Artificial Sequence Primer 28atcccgggca gcgagcacac agctagcaac tctt 34 29 32 DNA Artificial SequenceDescription of Artificial Sequence Primer 29 gcgagctcac tccctcacgcggttgacaat ct 32 30 25 DNA Artificial Sequence Description of ArtificialSequence Primer 30 tggcggcctc ggtcttcttc tcagt 25 31 33 DNA ArtificialSequence Description of Artificial Sequence Primer 31 atcccgggaggaagctgtca gggaagagat gga 33 32 34 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 32 gcgagctctg gccgtaaaat cagttgtggc gctt34 33 25 DNA Artificial Sequence Description of Artificial SequencePrimer 33 cagcgaagcc caatcgggat cagca 25 34 32 DNA Artificial SequenceDescription of Artificial Sequence Primer 34 atcccgggag gaggacttgcggaatgcaaa tc 32 35 33 DNA Artificial Sequence Description of ArtificialSequence Primer 35 gcgatatcca cctgcttcca ctctctactt atg 33 36 25 DNAArtificial Sequence Description of Artificial Sequence Primer 36gacacccgat tgagccggca agacg 25 37 33 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 37 atcccgggca ccagtcccgc ttagtgtgtg tgt 3338 31 DNA Artificial Sequence Description of Artificial Sequence Primer38 gcgagctctt gatgcgactc gctctctcga t 31 39 26 DNA Artificial SequenceDescription of Artificial Sequence Primer 39 cggcgagtgc agcagcttctagaacg 26 40 31 DNA Artificial Sequence Description of ArtificialSequence Primer 40 atcccgggta tcgatctgga gcccgttgca a 31 41 32 DNAArtificial Sequence Description of Artificial Sequence Primer 41gcgagctcct ccaaaggact ttgaaatata gc 32 42 33 DNA Artificial SequenceDescription of Artificial Sequence Primer 42 gatatcggaa gaagaatccaagggaatgcg gtt 33 43 33 DNA Artificial Sequence Description ofArtificial Sequence Primer 43 gcgagctcta tgcttccgtg ggaggagctt cac 33 4426 DNA Artificial Sequence Description of Artificial Sequence Primer 44ccggctgggt tgcctcagct tgcgca 26 45 26 DNA Artificial SequenceDescription of Artificial Sequence Primer 45 cgctccatcg aacctggtgcctttgc 26 46 32 DNA Artificial Sequence Description of ArtificialSequence Primer 46 atcccgggct cggaaggact gtgcattgtc ga 32 47 33 DNAArtificial Sequence Description of Artificial Sequence Primer 47gcgagctcgc agcagaagaa atccacttct ggt 33 48 26 DNA Artificial SequenceDescription of Artificial Sequence Primer 48 gggtgccggt tgatgcgagggtccag 26 49 29 DNA Artificial Sequence Description of ArtificialSequence Primer 49 atcccgggct gttgtgtaca gtctgtgga 29 50 32 DNAArtificial Sequence Description of Artificial Sequence Primer 50atcccgggct cacggagtaa aggccgtacc tt 32 51 30 DNA Artificial SequenceDescription of Artificial Sequence Primer 51 gcgctgcaga tttcatttggagaggacacg 30 52 35 DNA Artificial Sequence Description of ArtificialSequence Primer 52 cgcggccggc ctcagaagaa ctcgtcaaga aggcg 35 53 25 DNAArtificial Sequence Description of Artificial Sequence Primer 53gctgacacgc caagcctcgc tagtc 25 54 32 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 54 gcgagctcac tccctcacgc ggttgacaat ct 3255 34 DNA Artificial Sequence Description of Artificial Sequence Primer55 gcgagctctg gccgtaaaat cagttgtggc gctt 34 56 33 DNA ArtificialSequence Description of Artificial Sequence Primer 56 gcgatatccacctgcttcca ctctctactt atg 33 57 31 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 57 gcgagctctt gatgcgactc gctctctcga t 3158 32 DNA Artificial Sequence Description of Artificial Sequence Primer58 gcgagctcct ccaaaggact ttgaaatata gc 32 59 33 DNA Artificial SequenceDescription of Artificial Sequence Primer 59 gcgagctcta tgcttccgtgggaggagctt cac 33 60 33 DNA Artificial Sequence Description ofArtificial Sequence Primer 60 gcgagctcgc agcagaagaa atccacttct ggt 33 6132 DNA Artificial Sequence Description of Artificial Sequence Primer 61atcccgggct cacggagtaa aggccgtacc tt 32 62 34 DNA Artificial SequenceDescription of Artificial Sequence Primer 62 atcccgggca gcgagcacacagctagcaac tctt 34 63 32 DNA Artificial Sequence Description ofArtificial Sequence Primer 63 gcgagctcac tccctcacgc ggttgacaat ct 32 6426 DNA Artificial Sequence Description of Artificial Sequence Primer 64gcccgttgtg tcgcacgagt gtggga 26 65 25 DNA Artificial SequenceDescription of Artificial Sequence Primer 65 gccgctggac cagacctcgg aatgt25 66 25 DNA Artificial Sequence Description of Artificial SequencePrimer 66 gaggcagtca tgcaatcgac cccaa 25 67 26 DNA Artificial SequenceDescription of Artificial Sequence Primer 67 gcgaagccca atcgggatcagcagca 26 68 33 DNA Artificial Sequence Description of ArtificialSequence Primer 68 atcccgggca ccagtcccgc ttagtgtgtg tgt 33 69 31 DNAArtificial Sequence Description of Artificial Sequence Primer 69gcgagctctt gatgcgactc gctctctcga t 31 70 25 DNA Artificial SequenceDescription of Artificial Sequence Primer 70 cgcatcgcat ctggcgaact ttgtg25 71 26 DNA Artificial Sequence Description of Artificial SequencePrimer 71 cgtaccacga ttgctctagc gcacgt 26 72 35 DNA Artificial SequenceDescription of Artificial Sequence Primer 72 gcgatatcgg aagaagaatccaagggaatg cggtt 35 73 33 DNA Artificial Sequence Description ofArtificial Sequence Primer 73 gcgagctcta tgcttccgtg ggaggagctt cac 33 7434 DNA Artificial Sequence Description of Artificial Sequence Primer 74atcccgggca gcgagcacac agctagcaac tctt 34 75 32 DNA Artificial SequenceDescription of Artificial Sequence Primer 75 gcgagctcac tccctcacgcggttgacaat ct 32 76 26 DNA Artificial Sequence Description of ArtificialSequence Primer 76 gcccgttgtg tcgcacgagt gtggga 26 77 25 DNA ArtificialSequence Description of Artificial Sequence Primer 77 gccgctggaccagacctcgg aatgt 25 78 25 DNA Artificial Sequence Description ofArtificial Sequence Primer 78 gaggcagtca tgcaatcgac cccaa 25 79 26 DNAArtificial Sequence Description of Artificial Sequence Primer 79gcgaagccca atcgggatca gcagca 26

We claim:
 1. A transgenic plant cell transformed with a nucleic acidencoding a polypeptide, wherein the polypeptide is defined in SEQ IDNO:20.
 2. The transgenic plant cell of claim 1, wherein the nucleic acidcomprises a polynucleotide as defined in SEQ ID NO:12.
 3. A transgenicplant cell transformed with a nucleic acid encoding a polypeptide,wherein expression of the polypeptide in the plant cell results in theplant cell's increased tolerance to an environmental stress selectedfrom one or more of the group consisting of drought and temperature lessthan or equal to 0° C., as compared to a wild type variety of the plantcell; wherein the nucleic acid hybridizes under stringent conditions toat least one sequence from the group consisting of a sequence of SEQ IDNO:12 and the full-length complement of the sequence of SEQ ID NO:12;and wherein the stringent conditions comprise hybridization in a 6×sodium chloride/sodium citrate (SSC) solution at 65° C. and at least onewash in a 0.2×SSC, 0.1% SDS solution at 50° C.
 4. A transgenic plantcell transformed with a nucleic acid encoding a polypeptide having atleast 90% sequence identity with a polypeptide as defined in SEQ IDNO:20, wherein expression of the polypeptide in the plant cell resultsin the plant cell's increased tolerance to an environmental stressselected from one or more of the group consisting of drought andtemperature less than or equal to 0° C., as compared to a wild typevariety of the plant cell.
 5. The transgenic plant cell of any of claims1, 2, 3, or 4, wherein the plant is a monocot.
 6. The transgenic plantcell of any of claims 1, 2, 3, or 4, wherein the plant is a dicot. 7.The transgenic plant cell of any of claims 1, 2, 3, or 4, wherein theplant is selected from the group consisting of maize, wheat, rye, oat,triticale, rice, barley, soybean, peanut, cotton, rapeseed, canola,manihot, pepper, sunflower, tagetes, solanaceous plants, potato,tobacco, eggplant, tomato, Vicia species, pea, alfalfa, coffee, cacao,tea, Salix species, oil palm, coconut, and perennial grass.
 8. Atransgenic plant comprising the transgenic plant cell according to anyone of claims 1, 2, 3, or
 4. 9. A seed comprising the transgenic plantcell according to any one of claims 1, 2, 3, or
 4. 10. A seed producedby a transgenic plant comprising a plant cell according to any of claims1, 2, 3, or 4, wherein the seed comprises the nucleic acid encoding thepolypeptide, wherein the seed is true breeding for an increasedtolerance to an environmental stress as compared to a wild type varietyof the plant cell, and wherein the environmental stress is selected fromone or more of the group consisting of drought and temperature less thanor equal to 0° C.
 11. An isolated nucleic acid encoding a polypeptide,wherein the nucleic acid comprises a polynucleotide that encodes thepolypeptide as defined in SEQ ID NO:20.
 12. The nucleic acid of claim11, wherein the nucleic acid comprises the polynucleotide as defined inSEQ ID NO:12.
 13. An isolated nucleic acid encoding a polypeptide,wherein expression of the polypeptide in the plant cell results in theplant cell's increased tolerance to an environmental stress selectedfrom one or more of the group consisting of drought and temperature lessthan or equal to 0° C. as compared to a wild type variety of the plantcell; wherein the nucleic acid hybridizes under stringent conditions toat least one sequence from the group consisting of a sequence of SEQ IDNO:12 and the full-length complement of the sequence of SEQ ID NO:12;and wherein the stringent conditions comprise hybridization in a 6×sodium chloride/sodium citrate (SSC) solution at 65° C. and at least onewash in a 0.2×SSC, 0.1% SDS solution at 50° C.
 14. An isolated nucleicacid encoding a polypeptide having at least 90% sequence identity with apolypeptide as defined in SEQ ID NO:20, wherein expression of thepolypeptide in the plant cell results in the plant cell's increasedtolerance to an environmental stress selected from one or more of thegroup consisting of drought and temperature less than or equal to 0° C.,as compared to a wild type variety of the plant cell.
 15. A seedcomprising the isolated nucleic acid according to any one of claims 11,12, 13, or
 14. 16. An isolated recombinant expression vector comprisinga nucleic acid of any one of claims 11, 12, 13, or 14, whereinexpression of the polypeptide in a plant cell results in the plantcell's increased tolerance to an environmental stress as compared to awild type variety of the plant cell, and wherein the environmentalstress is selected from one or more of the group consisting of droughtand temperature less than or equal to 0° C.
 17. A method of producing atransgenic plant comprising a nucleic acid encoding a polypeptide,comprising, a. transforming a plant cell with the expression vector ofclaim 16; and b. generating from the plant cell a transgenic plant thatexpresses the polypeptide; wherein the polypeptide is defined in SEQ IDNO:20.
 18. The method of claim 17, wherein the expression vectorcomprises the polynucleotide as defined in SEQ ID NO:12.
 19. A method ofproducing a transgenic plant comprising a nucleic acid encoding apolypeptide, wherein expression of the polypeptide in the plant resultsin the plant's increased tolerance to an environmental stress ascompared to a wild type variety of the plant, comprising, a.transforming a plant cell with the expression vector of claim 16; and b.generating from the plant cell a transgenic plant that expresses thepolypeptide; wherein the nucleic acid hybridizes under stringentconditions to at least one sequence from the group consisting of asequence of SEQ ID NO:12 and the full-length complement of the sequenceof SEQ ID NO:12; wherein the stringent conditions comprise hybridizationin a 6× sodium chloride/sodium citrate (SSC) solution at 65° C. and atleast one wash in a 0.2×SSC, 0.1% SDS solution at 50° C.; and whereinthe environmental stress is selected from one or more of the groupconsisting of drought and temperature less than or equal to 0° C.
 20. Amethod of producing a transgenic plant comprising a nucleic acidencoding a polypeptide, wherein expression of the polypeptide in theplant results in the plant's increased tolerance to an environmentalstress as compared to a wild type variety of the plant, comprising, a.transforming a plant cell with the expression vector of claim 16; and b.generating from the plant cell a transgenic plant that expresses thepolypeptide; wherein the polypeptide has at least 90% sequence identitywith the polypeptide as defined in SEQ ID NO:20, and wherein theenvironmental stress is selected from one or more of the groupconsisting of drought and temperature less than or equal to 0° C.